Substituted tricyclic compounds as protein kinase inhibitors

ABSTRACT

Protein kinase inhibitors are disclosed having utility in the treatment of protein kinase-mediated diseases and conditions, such as cancer. The compounds of this invention have the following structure: 
                         
including steroisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein A is a ring moiety selected from:
 
                         
and wherein R1, R2, R3, X, Z, L1, Cycl1, L2 and Cycl2 are as defined herein. Also disclosed are compositions containing a compound of this invention, as well as methods relating to the use thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/965,313, filed Oct. 14, 2004, and is based on Provisional ApplicationNo. 60/608,529, entitled “Protein Kinase Inhibitors”, filed Sep. 9,2004; Provisional Application No. 60/511,486, entitled “Inhibitors ofc-kit and PDGFR Tyrosine Kinases,” filed Oct. 14, 2003; and ProvisionalApplication No. 60/511,489, entitled “Aurora-2 Kinase Inhibitors,” filedOct. 14, 2003, the disclosures of which are hereby incorporated byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Certain work disclosed herein was performed under grant numbers CA95031and CA88310 from the National Institutes of Health. The U.S. Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates, in general, to compounds that inhibitprotein kinase activity, and to compositions and methods relatedthereto.

2. Description of the Related Art

Cancer (and other hyperproliferative diseases) is characterized byuncontrolled cell proliferation. This loss of the normal control of cellproliferation often appears to occur as the result of genetic damage tocell pathways that control progress through the cell cycle. The cellcycle consists of DNA synthesis (S phase), cell division or mitosis (Mphase), and non-synthetic periods referred to as gap 1 (G1) and gap 2(G2). The M-phase is composed of mitosis and cytokinesis (separationinto two cells). All steps in the cell cycle are controlled by anorderly cascade of protein phosphorylation and several families ofprotein kinases are involved in carrying out these phosphorylationsteps. In addition, the activity of many protein kinases increases inhuman tumors compared to normal tissue and this increased activity canbe due to many factors, including increased levels of a kinase orchanges in expression of co-activators or inhibitory proteins.

Cells have proteins that govern the transition from one phase of thecell cycle to another. For example, the cyclins are a family of proteinswhose concentrations increase and decrease throughout the cell cycle.The cyclins turn on, at the appropriate time, different cyclin-dependentprotein kinases (CDKs) that phosphorylate substrates essential forprogression through the cell cycle. Activity of specific CDKs atspecific times is essential for both initiation and coordinated progressthrough the cell cycle. For example, CDK1 is the most prominent cellcycle regulator that orchestrates M-phase activities. However, a numberof other mitotic protein kinases that participate in M-phase have beenidentified, which include members of the polo, aurora, and NIMA(Never-In-Mitosis-A) families and kinases implicated in mitoticcheckpoints, mitotic exit, and cytokinesis.

Aurora kinases are a family of oncogenic serine/threonine kinases thatlocalize to the mitotic apparatus (centrosome, poles of the bipolarspindle, or midbody) and regulate completion of centrosome separation,bipolar spindle assembly and chromosome segregation. Three humanhomologs of aurora kinases have been identified (aurora-1, aurora-2 andaurora-3). They all share a highly conserved catalytic domain located inthe carboxyl terminus, but their amino terminal extensions are ofvariable lengths with no sequence similarity. The human aurora kinasesare expressed in proliferating cells and are also overexpressed innumerous tumor cell lines including breast, ovary, prostate, pancreas,and colon. Aurora-2 kinase acts as an oncogene and transforms both Rat1fibroblasts and mouse NIH3T3 cells in vitro, and aurora-2 transforms NIH3T3 cells grown as tumors in nude mice. Excess aurora-2 may drive cellsto aneuploidy (abnormal numbers of chromosomes) by accelerating the lossof tumor suppressor genes and/or amplifying oncogenes, events known tocontribute to cellular transformation. Cells with excess aurora-2 mayescape mitotic check points, which in turn can activate proto-oncogenesinappropriately. Up-regulation of aurora-2 has been demonstrated in anumber of pancreatic cancer cell lines. In additional, aurora-2 kinaseantisense oligonucleotide treatment has been shown to cause cell cyclearrest and increased apoptosis. Therefore, aurora-2 kinase is anattractive target for rational design of novel small molecule inhibitorsfor the treatment of cancer and other conditions.

C-kit is a transmembrane receptor belonging to the type 3 subgroup ofreceptor tyrosine kinases that also includes platelet-derived growthfactor receptor (PDGFR), colony-stimulating factor 1 receptor (CSF-1),and FMS-like tyrosine kinase (Flt-3). Gastrointestinal stromal tumors(GIST), which are the most common mesenchymal tumors of thegastrointestinal tract, have been demonstrated to frequentlyover-express c-kit. GISTs are thought to originate from the InterstitialCells of Cajal (ICCs) that play a role in the control of gut motility.ICCs express the c-kit proto-oncogene. When c-kit binds to its ligandstem cell factor (SCF) and dimerizes with another c-kit receptor,trans-autophosphorylation on tyrosines occurs and activates a number ofdownstream signaling pathways that lead to a proliferative response.These events are believed to contribute to the induction of GIST.

Other GISTs are associated with excess activity of platelet-derivedgrowth factor receptor A (PDGFR-A), which is considered a key player inthe new blood vessel formation necessary for tumors to grow beyond morethan a few millimeters. PDGFR-A is found in stroma and pericytes(support cells for blood vessels). PDGFR-A levels have been found to beincreased in a number of other tumor types.

Researchers have explored cancer treatment approaches that inhibittyrosine kinases and other proteins involved in uncontrolled signaltransduction. For example, the signal transduction inhibitors STI571,SU5614, CT52923 (herein HPK15) and PD1739 are known to inhibit theactivity of Bcr-Abl, c-kit and PDGFR tyrosine kinases. STI571 GLEEVEC™;a phenylaminopyrimidine) is a small molecule inhibitor currently used inthe clinic, which selectively blocks the BCR-ABL tyrosine kinase dimerin chronic myclogenous leukemia. However, GLEEVEC™ also has been shownto inhibit the c-kit and PDGFR tyrosine kinases and therefore may alsobe useful in tumors that over-express these receptors. Recent studies onpatients with metastatic GISTs treated with STI571 have shown decreasedtumor size on computed tomography and MRI and metabolic responsemeasured with 19-fluoro-desoxyglucose positron emission tomography(FDG-PET). However, two Phase I trials with STI571 at dose levels of 400mg or 600 mg per day showed a partial response in 54%, stable disease in34% and progressive disease in 12% of patients assessed at 1-3 months.These initial trials indicate that although a very good partial responsewas initially obtained, complete responses were quite rare, and patientseventually developed progressive disease. Recent studies showed that aparticular mutant (V560G) of c-kit is more sensitive to STI571, and amutant in the c-kit kinase domain (D816V) was resistant. Therefore, thedesign and development of novel inhibitors of mutant c-kit and/or ofPDGFR are needed for the treatment of GIST and other conditionsassociated with excess c-kit and/or PDGFR activity.

Quinazoline derivatives have been proposed for inhibiting protein kinaseactivity. For example, WO 96/09294, WO 96/33981 and EP 0837 063 describethe use of certain quinazoline compounds as receptor tyrosine kinaseinhibitors. In addition, WO 01/21596 proposes the use of quinazolinederivatives to inhibit aurora-2 kinase.

What remains needed, however, are additional and improved inhibitors ofprotein kinase activity, particularly inhibitors of aurora-2 kinase,c-kit and/or PDGFR-A kinase activity. The present invention fulfillsthese needs and offers other related advantages.

BRIEF SUMMARY OF THE INVENTION

The present invention is generally directed to compounds having thefollowing general structure (I):

including steroisomers, prodrugs and pharmaceutically acceptable saltsthereof, wherein A is a ring moiety selected from:

and wherein R₁, R₂, R₃, X, Z, L₁, Cycl₁, L₂ and Cycl₂ are as definedherein.

These compounds of the present invention have utility over a broad rangeof therapeutic applications, and may be used to treat diseases, such ascancer, that are mediated at least in part by protein kinase activity.Accordingly, in one aspect of the invention, the compounds describedherein are formulated as pharmaceutically acceptable compositions foradministration to a subject in need thereof.

In another aspect, the invention provides methods for treating orpreventing a protein kinase-mediated disease, such as cancer, whichmethod comprises administering to a patient in need of such a treatmenta therapeutically effective amount of a compound described herein or apharmaceutically acceptable composition comprising said compound. Incertain embodiments, the protein kinase-mediated disease is an aurora-2kinase-mediated disease or a c-kit-mediated disease.

Another aspect of the invention relates to inhibiting protein kinaseactivity in a biological sample, which method comprises contacting thebiological sample with a compound described herein, or apharmaceutically acceptable composition comprising said compound. Incertain embodiments, the protein kinase is aurora-2 kinase, PDGFR-a orc-kit kinase.

Another aspect of this invention relates to a method of inhibitingprotein kinase activity in a patient, which method comprisesadministering to the patient a compound described herein or apharmaceutically acceptable composition comprising said compound. Incertain embodiments, the protein kinase is aurora-2 kinase or c-kitkinase.

These and other aspects of the invention will be apparent upon referenceto the following detailed description and attached figures. To that end,certain patent and other documents are cited herein to more specificallyset forth various aspects of this invention. Each of these documents ishereby incorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays the general structures of illustrative compounds of thepresent invention.

FIG. 2 displays structure-based sequence alignments in the Clustal Xprogram (multiple alignment program, EMBL-EBI, UK) of the catalyticprotein kinase domains of aurora-2 (ARK1); SEQ ID NO. 1, aurora-1(ARK2); SEQ ID NO. 2, bovine cAMP-dependent PK (1CDK): SEQ ID NO. 3,murine cAMP-dependent PK (1APM); SEQ ID NO. 4, and C. elegans twitchinkinase (1KOA); SEQ ID NO. 5. Black bars: α-helices (α1-α11); gray bars:β-sheets (β1-β11); shaded and *: identical residues; :: highly conservedresidues; and *: similar residues.

FIG. 3 displays the homology model of aurora-2 kinase. Secondarystructural elements include α-helix, β-sheet, coil, and turns.

FIG. 4 displays the structures of the ATP analog (AMP-PNP) and S/Tkinase inhibitors (staurosporine, H-89, H-8, H-7, KN-93, ML-7, and6,7-dimethoxyquinazoline) evaluated for inhibitory activities againstaurora-2 kinase.

FIG. 5 shows the superposed structures of staurosporine,6,7-dimethoxyquinazoline, H-89, and AMP-PNP docked into the ATP-bindingpocket of aurora-2. The enzyme active site is clipped.

FIG. 6 shows the purine, quinazoline, isoquinazoline and indole ringtemplates used in LUDI search.

FIG. 7A displays structures of illustrative pyrimido[4,5-b]indoles. FIG.7B displays structures of illustrative benzofuranopyrimidines. FIG. 7Cdisplays structures of illustrative benzothieno[3,2-d]pyrimidone. FIG.7D displays structures of illustrative 6,7-dimethoxyquinazolines.

FIG. 8 shows schematic synthetic methods for making illustrativecompounds of the invention.

FIG. 9 shows the schematic synthesis of compounds HPK 16 and HPK 62.

FIG. 10 is a bar graph showing inhibition of aurora-2 kinase byillustrative compounds (20 μM) in an in vitro assay.

FIG. 11 graphs aurora-2 kinase inhibition by five compounds at differentconcentrations to determine the concentration providing 50% inhibition(IC₅₀).

FIG. 12 displays the general structures of further illustrativeinventive compounds.

FIG. 13 displays structure-based sequence alignments in the Clustal Xprogram (multiple alignment program, EMBL-EBI, UK) of the catalyticprotein kinase domains of c-kit; SEQ ID NO. 6, PDGDR-α; SEQ ID NO. 7,PDGFR-β; SEQ ID NO. 8, FGFr1 SEQ ID NO. 9, VEGFR2; SEQ ID NO. 10 andBCR-ABL; SEQ ID NO. 11. Shaded and * are identical residues; “::” arehighly conserved residues; and • are similar residues. The N-terminaland C-terminal extensions of c-kit are not included in the modeling.

FIG. 14 displays the homology model of c-kit bound compound 1 dockedinto the ATP binding site.

FIGS. 15A and 15B are molecular models of the c-kit binding site withtwo different prior art compounds, CT52923 and STI571, respectively.

FIG. 16 shows the purine, quinazoline, isoquinazoline,pyrimido[4,5-b]indoles, benzothieno[3,2-d], benzofuranopyrimidines andindole ring structures used in the LUDI search.

FIG. 17 shows the structures of novel4-piprazinylpyrimido[4,5-b]indoles, benzothieno[3,2-d],benzofuranopyrimidines and quinazoline inhibitors designed as c-kittyrosine kinase inhibitors.

FIGS. 18A and 18B show molecular models of the c-kit kinase active sitepocket containing compounds 3 and 1, respectively.

FIG. 19 shows a molecular model developed with FlexX software. It showsdocking and overlay of compound 3 and STI571 within the c-kit kinaseactive site pocket.

FIG. 20 depicts the synthesis of seven illustrative compounds.

FIG. 21 summarizes the preparation of intermediates 1c and 1d.

FIGS. 22A, 22B, and 22C display graphically the results of in vitrocytotoxicity testing of GIST882, MIAPaCa-2 and PANC-1 cell lines,respectively.

FIG. 23 shows the effects of compound (II-2-6) on cell cycledistribution of the MIA PaCa-2 pancreatic cancer cell line.

FIG. 24 shows the effects of compound (II-2-6) on cell proliferation ofthe MIA PaCa-2 pancreatic cancer cell line.

FIGS. 25A and 25B show the effects of compound (II-2-6) on in vitrocytotoxicity of the MIA PaCa-2 pancreatic cancer cell line.

FIGS. 26A and 26B and 26C show the effects of compound (II-2-6) on invitro cytotoxicity of colon, breast, ovarian and pancreatic cancer celllines.

FIG. 27 shows the kinase inhibitory activity of compound (II-2-6)against multiple protein kinases.

FIGS. 28A and 28B show the results of phosphorylation assays for c-kitand PDGFR-a, respectively.

FIG. 29 shows the inhibitory activity of illustrative compounds in theGIST cell line, GIST882.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to compounds useful asprotein kinase inhibitors and to compositions and methods relatingthereto. Such compounds of the invention have the following structure(I):

including steroisomers, prodrugs and pharmaceutically acceptable saltsthereof, wherein A is a ring moiety selected from:

and wherein:

X is NH, S or O;

Z is CH or N;

R₁ and R₂ are the same or different and are independently hydrogen,hydroxyl, halo, —CN, —NO₂, —NH₂, —R, —OR, —SCH₃, —CF₃, —C(═O)OR or—OC(═O)R, where R is alkyl or substituted alkyl;

R₃ is hydrogen, —NH₂, alkyl, —CN, or —NO₂, or R₃ is -L₃-Cycl₃ wherein L₃is a direct bond, —S— or —NH—, and Cycl₃ is a carbocycle, substitutedcarbocycle, heterocycle or substituted heterocycle;

L₁ is a direct bond, —NR′—, —OC(═S)NH— or —NHC(═S)O—; wherein R′ is H oralkyl;

Cycl₁ is optional and, when present, is a carbocycle, substitutedcarbocycle, heterocycle or substituted heterocycle;

L₂ is a direct bond or —C(═S)NH—, —NHC(═S)—, —NHC(═S)NH—, —C(═O)NH—,—NHC(═O)—, —NHC(═O)NH—, —(CH₂)_(n)—, —NH(CH₂)_(n)—, —(CH₂)_(n)NH—,—NH(CH₂)_(n)NH—, —C(═S)NH(CH₂)_(n)—, —NHC(═S)(CH₂)_(n)—,—(CH₂)_(n)C(═S)NH(CH₂)_(n)—, —(CH₂)_(n)NHC(═S)(CH₂)_(n)—, —NHC(═O)—,—S(═O)₂—, —S(═O)₂NH—, —NHS(═O)₂—, wherein n is, at each occurrence thesame or different and independently 1, 2, 3 or 4; and

Cycl₂ is a carbocycle, substituted carbocycle, heterocycle orsubstituted heterocycle.

Unless otherwise stated the following terms used in the specificationand claims have the meanings discussed below:

“Alkyl” refers to a saturated straight or branched hydrocarbon radicalof one to six carbon atoms, preferably one to four carbon atoms, e.g.,methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl, tert-butyl, pentyl,hexyl, and the like, preferably methyl, ethyl, propyl, or 2-propyl.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, —CH₂-cyclohexyl, andthe like; while unsaturated cyclic alkyls include cyclopentenyl,cyclohexenyl, —CH₂-cyclohexenyl, and the like. Cyclic alkyls are alsoreferred to herein as a “cycloalkyl.” Unsaturated alkyls contain atleast one double or triple bond between adjacent carbon atoms (referredto as an “alkenyl” or “alkynyl”, respectively.) Representative straightchain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; whilerepresentative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl,3-methyl-1-butynyl, and the like.

“Alkylene” means a linear saturated divalent hydrocarbon radical of oneto six carbon atoms or a branched saturated divalent hydrocarbon radicalof three to six carbon atoms, e.g., methylene, ethylene,2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene,and the like, preferably methylene, ethylene, or propylene.

“Cycloalkyl” refers to a saturated cyclic hydrocarbon radical of threeto eight carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl.

“Alkoxy” means a radical —OR_(a) where R_(a) is an alkyl as definedabove, e.g., methoxy, ethoxy, propoxy, butoxy and the like.

“Halo” means fluoro, chloro, bromo, or iodo, preferably fluoro andchloro.

“Haloalkyl” means alkyl substituted with one or more, preferably one,two or three, same or different halo atoms, e.g., —CH₂Cl, —CF₃, —CH₂CF₃,—CH₂CCl₃, and the like.

“Haloalkoxy” means a radical —OR_(b) where R_(b) is an haloalkyl asdefined above, e.g., trifluoromethoxy, trichloroethoxy,2,2-dichloropropoxy, and the like.

“Acyl” means a radical —C(O)R_(c) where R_(c) is hydrogen, alkyl, orhaloalkyl as defined herein, e.g., formyl, acetyl, trifluoroacetyl,butanoyl, and the like.

“Aryl” refers to an all-carbon monocyclic or fused-ring polycyclic(i.e., rings which share adjacent pairs of carbon atoms) groups of 6 to12 carbon atoms having a completely conjugated pi-electron system.Examples, without limitation, of aryl groups are phenyl, naphthyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the aryl group is substituted with one or more, morepreferably one, two or three, even more preferably one or twosubstituents independently selected from the group consisting of alkyl,haloalkyl, halo, hydroxy, alkoxy, mercapto, alkylthio, cyano, acyl,nitro, phenoxy, heteroaryl, heteroaryloxy, haloalkyl, haloalkoxy,carboxy, alkoxycarbonyl, amino, alkylamino or dialkylamino.

“Heteroaryl” refers to a monocyclic or fused ring (i.e., rings whichshare an adjacent pair of atoms) group of 5 to 12 ring atoms containingone, two, three or four ring heteroatoms selected from N, O, or S, theremaining ring atoms being C, and, in addition, having a completelyconjugated pi-electron system. Examples, without limitation, ofunsubstituted heteroaryl groups are pyrrole, furan, thiophene,imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline,isoquinoline, purine, triazole, tetrazole, triazine, and carbazole. Theheteroaryl group may be substituted or unsubstituted. When substituted,the heteroaryl group is substituted with one or more, more preferablyone, two or three, even more preferably one or two substituentsindependently selected from the group consisting of alkyl, haloalkyl,halo, hydroxy, alkoxy, mercapto, alkylthio, cyano, acyl, nitro,haloalkyl, haloalkoxy, carboxy, alkoxycarbonyl, amino, alkylamino ordialkylamino.

“Carbocycle” refers to an aliphatic ring system having 3 to 14 ringatoms. The term “carbocycle”, whether saturated or partiallyunsaturated, also refers to rings that are optionally substituted. Theterm “carbocycle” also includes aliphatic rings that are fused to one ormore aromatic or nonaromatic rings, such as in a decahydronaphthyl ortetrahydronaphthyl, where the radical or point of attachment is on thealiphatic ring.

“Heterocycle” refers to a saturated cyclic ring system having 3 to 14ring atoms in which one, two or three ring atoms are heteroatomsselected from N, O, or S(O)_(m) (where m is an integer from 0 to 2), theremaining ring atoms being C, where one or two C atoms may optionally bereplaced by a carbonyl group. The heterocyclyl ring may be optionallysubstituted independently with one or more, preferably one, two, orthree substituents selected from alkyl (wherein the alkyl may beoptionally substituted with one or two substituents independentlyselected from carboxy or ester group), haloalkyl, cycloalkylamino,cycloalkylalkyl, cycloalkylaminoalkyl, cycloalkylalkylaminoalkyl,cyanoalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, alkylamino,dialkylamino, hydroxyalkyl, carboxyalkyl, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, saturated or unsaturated heterocycloamino, saturated orunsaturated heterocycloaminoalkyl, and —COR_(d) (where R_(d) is alkyl).More specifically the term heterocyclyl includes, but is not limited to,tetrahydropyranyl, 2,2-dimethyl-1,3-dioxolane, piperidino,N-methylpiperidin-3-yl, piperazino, N-methylpyrrolidin-3-yl,pyrrolidino, morpholino, 4-cyclopropylmethylpiperazino, thiomorpholino,thiomorpholino-1-oxide, thiomorpholino-1,1-dioxide,4-ethyloxycarbonylpiperazino, 3-oxopiperazino, 2-imidazolidone,2-pyrrolidinone, 2-oxohomopiperazino, tetrahydropyrimidin-2-one, and thederivatives thereof. In certain embodiments, the heterocycle group isoptionally substituted with one or two substituents independentlyselected from halo, alkyl, alkyl substituted with carboxy, ester,hydroxy, alkylamino, saturated or unsaturated heterocycloamino,saturated or unsaturated heterocycloaminoalkyl, or dialkylamino.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “heterocyclic group optionallysubstituted with an alkyl group” means that the alkyl may but need notbe present, and the description includes situations where theheterocycle group is substituted with an alkyl group and situationswhere the heterocycle group is not substituted with the alkyl group.

Lastly, the term “substituted” as used herein means any of the abovegroups (e.g., alkyl, aryl, heteroaryl, carbocycle, heterocycle, etc.)wherein at least one hydrogen atom is replaced with a substituent. Inthe case of an oxo substituent (“═O”) two hydrogen atoms are replaced.“Substituents” within the context of this invention include halogen,hydroxy, oxo, cyano, nitro, amino, alkylamino, dialkylamino, alkyl,alkoxy, thioalkyl, haloalkyl, hydroxyalkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,—NR_(e)R_(f), —NR_(e)C(═O)R_(f), —NR_(e)C(═O)NR_(e)R_(f),—NR_(e)C(═O)OR_(f)—NR_(e)SO₂R_(f), —OR_(e), —C(═O)R_(e)—C(═O)OR_(e),—C(═O)NR_(e)R_(f), OC(═O)NR_(e)R_(f), —SH, —SR_(e), —SOR_(e),—S(═O)₂R_(e), —OS(═O)₂R_(e), —S(═O)₂OR_(e), wherein R_(e) and R_(f) arethe same or different and independently hydrogen, alkyl, haloalkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl or substituted heterocyclealkyl.

In one embodiment of the invention, ring moiety A of structure (I) is asshown above in (I-A), and the compounds have the following structure(II):

In another embodiment, the present invention provides more specificcompounds of structure (II) wherein La is a direct bond, and thecompounds have the following structure (II-1):

In a more specific aspect of structure II-1 above, Cycl₁ is aheterocycle or substituted heterocycle.

In a more specific aspect of structure II-1 above, Cycl₁ is aheterocycle or substituted heterocycle, and the compounds have thefollowing structures (II-2) to (II-5):

In a more specific aspect of structure (II-2), L₂ is either —C(═S)NH— or—C(═S)NHCH₂—, and the compounds have the structures (II-2-1) and(II-2-2), respectively:

In more specific aspects of structure (II-2-1) and (II-2-2) above, X isNH and Z is CH.

In more specific aspects of structure (II-2-1) and (II-2-2) above, L₂ iseither —C(═S)NH— or —C(═S)NHCH₂—.

In more specific aspects of structure (II-2-1) and (II-2-2) above, X isNH, Z is CH, L₂ is either —C(═S)NH— or —C(═S)NHCH₂—, and the compoundshave the following structures (II-2-3) and (II-2-4), respectively:

In more specific aspects of structures (II-2-3) and (II-2-4) above,Cycl₂ is selected from:

In more specific aspects of structure (II-2-3) and (II-2-4), R₁ and R₂are selected from —OCH₃, —OH, —Cl, —CF₃, or —OC(═O)CH₃, and R₃ isselected from hydrogen or —NH₂.

In a more specific aspect of structure (II-2-3), Cycl₂ is a substitutedcarbocyle.

In a more specific aspect of structure (II-2-3), Cycl₂ is a substitutedcarbocyle, and the compounds have the following structure (II-2-5)below:

In a more specific aspect of structure (II-2-5), R₁ and R₂ are methoxy,R₃ is H, and the compound has the following structure (II-2-6):

In a more specific aspect of structure (II-2-4) above, R₁ and R₂ aremethoxy and R₃ is hydrogen.

In a more specific aspect of structure (II-2-4) above, R₁ and R₂ aremethoxy, R₃ is hydrogen, and Cycl₂ is:

and the compound has the following structure (II-2-7):

In more specific aspects of structure (II-3), Z is CH and X is NH, andthe compounds have the following structure (II-3-1):

In more specific aspects of structure (II-3-1), R₁ and R₂ are methoxyand R₃ is hydrogen, and the compounds have the following structure(II-3-2):

In a more specific aspect of structure (II-3-2) above, L₂ is —NHC(═S)NH—or —NHC(═O)— and Cycl₂ is:

In a more specific aspect of structure (II-1) above, Cycl₁ is notpresent, L₂ is a direct bond, and Cycl₂ is a heterocycle or substitutedheterocycle.

In a more specific aspect of structure (II-1) above, Cycl₁ is notpresent, L₂ is a direct bond, Cycl₂ is a substituted heterocycle, andthe compounds have the following structure (II-3-3) below:

In a more specific aspect structure (II-4) above, Z is CH and X is NH,and the compounds have the following structure (II-4-1):

In a more specific aspect structure (II-4-1) above, R₁ and R₂ aremethoxy and R₃ is hydrogen, and the compounds have the followingstructure (II-4-2):

In more specific aspects of structure (II-4-2) above, L₂ is —NHC(═O)NH—,—NHC(═O)— or —HNC(═S)NH—, and Cycl₂ is selected from:

where w is

In a more specific aspect of structure (II-I) above, Cycl₁ is notpresent, L₂ is a direct bond, and Cycl₂ is a heterocycle or substitutedheterocycle.

In a more specific aspect of structure (II-I) above, Z is CH, X is NH,Cycl₁ is not present, L2 is a direct bond and Cycl₂ is a heterocycle orsubstituted heterocycle.

In a more specific aspect of structure (II-I) above, Z is CH, X is NH,Cycl₁ is not present, L2 is a direct bond and Cycl₂ is a heterocycle orsubstituted heterocycle, and the compounds have the following structure(II-4-3) below:

In a more specific aspect of structure (II-4-3) above, w is —NO₂.

In a more specific aspect of structure (II-5) above, Z is CH and X isNH, and the compounds have the following structure (II-5-1):

In a more specific aspect of structure (II-5-1) above, R₁ and R₂ aremethoxy and R₃ is hydrogen, and the compounds have the followingstructure (II-5-2):

In a more specific aspect of structure (II-5-2) above, L₂ is —NHC(═O)—and Cycl₂ is a carbocycle.

In a more specific aspect of structure (II-5-2) above, L₂ is —NHC(═O)—and Cycl₂ is phenyl.

In another embodiment, the present invention provides compounds ofstructure (II) above wherein L₁ is —NH— or —OC(═S)NH—, and the compoundshave the following structures (II-6) and (II-7), respectively:

In a more specific aspect of structure (II-6), Cycl₁ is a carbocycle orheterocycle, and the compounds have the following structures (II-6-1) to(II-6-6):

In more specific aspects of structure (II-6-1) to (II-6-6), Z is CH, Xis NH and the compounds have the following structures (II-6-7) to(II-6-12):

In a more specific aspect of structure (II-6-7) above, R₁ and R₂ areboth methoxy, and R₃ is hydrogen.

In a more specific aspect of structure (II-6-7) above, R₁ and R₂ areboth methoxy, R₃ is hydrogen, and L₂ is —NHCH₂—, —NHC(═O)— or —NH—.

In a more specific aspect of structure (II-6-7) above, R₁ and R₂ areboth methoxy, R₃ is hydrogen, L₂ is —NHCH₂—, —NHC(═O)— or —NH—, andCycl₂ is:

where w is

In a more specific aspect of structure (II-6-8) above, R₁ and R₂ areboth methoxy, and R₃ is hydrogen.

In a more specific aspect of structure (II-6-8) above, R₁ and R₂ areboth methoxy, R₃ is hydrogen, and L₂ is —NHCH₂—, —NHC(═S)NH—, —NHC(═O)—or —NH—.

In a more specific aspect of structure (II-6-8) above, R₁ and R₂ areboth methoxy, R₃ is hydrogen, L₂ is —NHCH₂—, —NHC(═S)NH—, —NHC(═O)— or—NH—, and Cycl₂ is:

where w is

In a more specific aspect of structure (II-6-9) above, R₁ and R₂ areboth methoxy, and R₃ is hydrogen.

In a more specific aspect of structure (II-6-9) above, L₂ is —NHC(═O)—.

In a more specific aspect of structure (II-6-9) above, R₁ and R₂ areboth methoxy, R₃ is hydrogen, and L₂ is —NHC(═O)—.

In a more specific aspect of structure (II-6-9) above, Cycl₂ is phenyl.

In a more specific aspect of structure (II-6-9) above, R₁ and R₂ areboth methoxy, R₃ is hydrogen, L₂ is —NHC(═O)—, and Cycl₂ is phenyl.

In more specific aspects of structures (II-6-10), (II-6-11) and(II-6-12) above, R₁ and R₂ are both methoxy, and R₃ is hydrogen or —NH₂.

In more specific aspects of structures (II-6-10), (II-6-11) and(II-6-12) above, L₂ is —NHC(═S)NH—, —NHC(═S)— or —S(═O)₂—.

In more specific aspects of structures (II-6-10), (II-6-11) and(II-6-12) above, Cycl₂ is:

wherein w is —NH₂, —NO₂ or:

In more specific aspects of structures (II-6-10), (II-6-11) and(II-6-12) above, R₁ and R₂ are both methoxy, R₃ is hydrogen or —NH₂, andL₂ is —NHC(═S)NH—, —NHC(═S)— or —S(═O)₂—.

In more specific aspects of structure (II-6-10), (II-6-11) and (II-6-12)above, R₁ and R₂ are both methoxy, R₃ is hydrogen or —NH₂, L₂ is—NHC(═S)NH—, —NHC(═S)— or —S(═O)₂—, and Cycl₂ is:

wherein w is —NH₂, —NO₂ or:

In another embodiment relating to structure (I) of the invention, ringmoiety A is as shown above in (I-B), and the compounds having thefollowing structure (III):

In another embodiment, the present invention provides compounds ofstructure (III) in which L₁ is a direct bond and having structure(III-1) below:

In a more specific aspect of structure (III-1) above, Cycl₁ is aheterocycle.

In a more specific aspect of structure (III-1) above, Cycl₁ is aheterocycle, and the compounds have the structure (III-1-1) below:

In a more specific aspect of structure (III-1-1), R₁ and R₂ are selectedfrom hydrogen, methoxy or hydroxyl, and R3 is selected from hydrogen or—NH₂, and the compounds have the following structure (III-1-2) below:

In a more specific aspect of structure (III-1-2) above, X is S, O or NH,Z is CH or N.

In a more specific aspect of structure (III-1-2) above, R₁, R₂ and R₃are hydrogen.

In a more specific aspect of structure (III-1-2) above, X is S, O or NH,Z is CH or N, and R₁, R₂ and R₃ are hydrogen.

In a more specific aspect of structure (III-1-2) above, L₂ is selectedfrom —C(═S)NH—, —C(═S)—, —C(═S)NHCH₂— or —CH₂—.

In a more specific aspect of structure (III-1-2) Cycl₂ is selected from:

where w is

In a more specific aspect of structure (III-1-2), X is S, O or NH, Z isCH or N, R₁, R₂ and R₃ are hydrogen, and L₂ is selected from —C(═S)NH—,—C(═S)—, —C(═S)NHCH₂— or —CH₂—.

In a more specific aspect of structure (III-1-2), X is S, O or NH, Z isCH or N, R₁, R₂ and R₃ are hydrogen, L₂ is selected from —C(═S)NH—,—C(═S)—, —C(═S)NHCH₂— or —CH₂—, and Cycl₂ is selected from:

where w is

In a more specific aspect of structure (III-1-2) above, Z is CH and X isO.

In a more specific aspect of structure (III-1-2) above, Z is CH, X is O,and L₂ is —C(═S)NHCH₂—.

In a more specific aspect of structure (III-1-2) above, Z is CH, X is O,L₂ is —C(═S)NHCH₂—, and Cycl₂ is:

and the compound has the following structure (III-1-3):

In a more specific aspect of structure (III-1-2) above, Z is N and X isS.

In a more specific aspect of structure (III-1-2) above, Z is N, X is Sand R₁, R₂ and R₃ are hydrogen.

In a more specific aspect of structure (III-1-2) above, Z is N, X is S,R₁, R₂ and R₃ are hydrogen, and L₂ is —C(═S)NHCH₂—.

In a more specific aspect of structure (III-1-2) above, Z is N, X is S,R₁, R₂ and R₃ are hydrogen, L₂ is —C(═S)NHCH₂—, and Cycl₂ is:

and the compound has the following structure (III-1-4):

In a more specific aspect of structure (III-1-2) above, Z is CH and X isO.

In a more specific aspect of structure (III-1-2) above, Z is CH and X isO, and R₁, R₂ and R₃ are hydrogen.

In a more specific aspect of structure (III-1-2) above, Z is CH and X isO, R₁, R₂ and R₃ are hydrogen, and L₂ is —C(═S)NH—.

In a more specific aspect of structure (III-1-2) above, Z is CH, X is O,R₁, R₂ and R₃ are hydrogen, L₂ is —C(═S)NH—, and Cycl₂ is:

where w is

and the compound has the following structure (III-1-5):

In another embodiment relating to compounds of structure (III) above, L₁is —NH— or —OC(═S)NH—, and the compounds have structures (III-2) and(III-3) below:

In a more specific aspect of structure (III-2), R₁, R₂ and R₃ arehydrogen, and the compounds have structures (III-2-1) and (III-2-2)below:

In more specific aspects of structures (III-2-1) and (III-2-2) above,Cycl₁ is selected from:

In more specific aspects of structures (III-2-1) and (III-2-2) above, L₂is selected from —NHC(═S)NH—, —NHC(═O)—, —NH—, or —NHCH₂—.

In more specific aspects of structures (III-2-1) and (III-2-2) above, L₂is selected from —NHC(═S)NH—, —NHC(═O)—, —NH—, or —NHCH₂—, and Cycl₂ isselected from a carbocycle or substituted carbocycle.

In more specific aspects of structures (III-2-1) and (III-2-2) above, L₂is selected from —NHC(═S)NH—, —NHC(═O)—, —NH—, or —NHCH₂—, and Cycl₂ isselected from:

where w is

In another embodiment relating to structure (I), ring moiety A is asshown above in (I-C), and the compounds have the following structure(IV):

In another embodiment, the present invention provides compounds ofstructure (IV) wherein L₁ is a direct bond, and the compounds have thefollowing structure (IV-1):

In another embodiment relating to structure (IV-1), Cycl₁ is aheterocycle or substituted heterocycle.

In another embodiment relating to structure (IV-1), Cycl₁ is aheterocycle, and the compounds have the structure (IV-1-1) below:

In a more specific aspect of structure (IV-1-1), R₁ and R₂ are bothmethoxy, and R₃ is hydrogen.

In a more specific aspect of structure (IV-1-1), R₁ and R₂ are bothmethoxy, R₃ is hydrogen, and the compounds have the structure (IV-1-2)below:

In a more specific aspect of structures (IV-1-2), L₂ is —C(═S)NH—.

In a more specific aspect of structure (IV-1-2), L₂ is —C(═S)NH— andCycl₂ is.

where w is

and the compound has the following structure (IV-1-3):

In another embodiment relating to compounds of structure (IV) above, L₁is —NH—, and these compounds of the invention have the structures IV-2below:

In a more specific aspect of structure (IV-2), R₁ and R₂ are both frommethoxy and R₃ is hydrogen.

In a more specific aspect of structure (IV-2), R₁ and R₂ are bothmethoxy, R₃ is hydrogen, and Cycl₁ is a heterocycle or substitutedheterocycle.

In a more specific aspect of structure (IV-2), R₁ and R₂ are bothmethoxy, R₃ is hydrogen, and Cycl₁ is a heterocycle, and the compoundshave the structure (IV-2-1) below:

In a more specific aspect of structures (IV-2-1), L₂ is selected from—NHC(═S)NH—, —NH— or —NHCH₂—.

In a more specific aspect of structures (IV-2-1), L₂ is not —NHC(═O)—.

In a more specific aspect of structures (IV-2-1), L₂ is selected from—NHC(═S)NH—, —NH— or —NHCH₂— and Cycl₂ is selected from:

wherein w is L₄-Cycl₄, wherein L₄ is selected from —S(═O)₂NH—,—NHC(═S)NHCH₂—, —NHCH₂— or —NHC(═S)NH—, and wherein Cycl₄ is:

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers”. Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers”. Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers”. When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog (Cahn, R., Ingold, C., and Prelog, V. Angew. Chem.78:413-47, 1966; Angew. Chem. Internat. Ed. Eng. 5:385-415, 511, 1966),or by the manner in which the molecule rotates the plane of polarizedlight and designated as dextrorotatory or levorotatory (i.e., as (+) or(−)-isomers respectively). A chiral compound can exist as eitherindividual enantiomer or as a mixture thereof. A mixture containingequal proportions of the enantiomers is called a “racemic mixture”.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see discussion in Ch. 4 of ADVANCED ORGANICCHEMISTRY, 4^(th) edition, March, J., John Wiley and Sons, New YorkCity, 1992).

The compounds of the present invention may exhibit the phenomena oftautomerism and structural isomerism. For example, the compoundsdescribed herein may adopt an E or a Z configuration about the doublebond connecting the 2-indolinone moiety to the pyrrole moiety or theymay be a mixture of E and Z. This invention encompasses any tautomericor structural isomeric form and mixtures thereof which possess theability to modulate aurora-2 kinase activity and is not limited to, anyone tautomeric or structural isomeric form.

It is contemplated that a compound of the present invention would bemetabolized by enzymes in the body of the organism such as human beingto generate a metabolite that can modulate the activity of the proteinkinases. Such metabolites are within the scope of the present invention.

The compounds of this invention may be made by one skilled in this fieldaccording to the following general reaction schemes, as well as by themore detailed procedures set forth in the Examples.

Substituted tricyclic pyrimido[5,4-b]indole compounds (having structure(I) above where ring moiety A is (I-A)), benzothieno[3,2-d,benzofurano-pyrimidine compounds (having structure (I) above where ringmoiety A is (I-B)) and quinazoline compounds (having structure (I) abovewhere ring moiety A is (I-C)) can be prepared as outlined generally inScheme 1 below.

Chlorination of (un)substituted 6-membered aromatic moieties can becarried out in the presence of sulfuryl chloride at about 0° C. The4-chloro-(un)substituted benzene (2) can be nitrated to obtain1-chloro-(un)substituted-2-nitrobenzene (3) with fuming nitric acid,preferably without the temperature exceeding about 25° C. Ethyl2-cyano-2-(un)substituted-2-nitrophenyl)acetate (4) can be prepared byreacting compound 3 with ethylcyanoacetate in the presence ofpotassium-tert-butoxide in THF (yielded compound 4 at 23%). Further theyields can be optimized at this stage by reacting compound 3 in thepresence of K₂CO₃ in DMF at a temperature of about 155° C. for 6 hoursto give the ethylcyano ester in high yield. Reduction of ester 4, can becarried out with excess of Zn dust (4-6 eq) using known conditions togive an ethyl 2-amino-5,6-dimethoxy-1H-indole-3-carboxylate (5) withoutan N-hydroxy side product.

Both the benzofuranopyrimidine and the benzothieno[3,2-d]pyrimidones(I-B) can be prepared by alkylation of (un)substituted-2-cyanophenol(11) with methyl bromoacetate followed by cyclization in the presence ofNaH and DMSO, to give the benzofuran (13) in quantitative yields.Similarly, treatment of 2-chloronicotinonitrile (14) with ethylthioglycolate in the presence of NaH/DMSO gives the cyclic methyl ester(15) in good yields. Cyclization to knowndihydro-4H-pyrimido[4,5-b]indoles or the congeners;3H-Benzofurano[3,2-d]pyrimid-4-one and 3H-thieno[3,2-d]pyrimid-4-one tothe corresponding pyrimido[4,5-b]indol-4-ones respectively, can beperformed by heating at about 155 to 220° C. in formamide and catalyticsodium methoxide.

The dihydro-pyrimidines can be converted to 4-chlorides (7) in goodyields with Vilsmeier's reagent (oxalyl chloride/DMF) or thionylchlorideand/or POCl₃ in dioxane solvent. The 4-chlorides can be utilized inpreparing either 4-amino or 4-piprazine substituted tricyclic analoguesas outlined in Scheme 1. Condensation of 4-chlorides can then be carriedout with substituted aromatic amines to provide various compounds of theinvention. The reaction can be carried out in refluxing lower alcohol orDMA with a catalytic amount of dry HCl gas. Similarly the 4-chloridescan be reacted with piprazine in the presence of pyridine at refluxtemperature to give compound 8 in good yields. The quinazolines offormula I-C can be prepared by reacting (un)substituted anthranilic acidand formamide at 190° C. to give the dihydro-quinazolines. Under similarconditions to that of tricyclic-dihydropyrimido-indoles, the 4-chlorideanalogues of quinazolines can be prepared. The substitutent at the R₃position can be obtained by reacting either cyclic ethyl or methylesters in presence of cyanoacetamide and dry HCl to give the guanidineanalogues 16 and 17. These compounds can be cyclized to 3-substitutedtricyclic pyrimidine in presence of aqueous NaOH.

Certain intermediates that can be utilized in the preparation of targetcompounds are outlined in Scheme 2. The variously substituted aromaticamines can be treated with thiophosgene in CH₂Cl₂/TEA to give thioureaanalogue 20 in moderate yields. The compounds of formula I having4-substituted piprazine analogues can be prepared by reacting compound20 in the presence of TEA or pyridine. Similarly, 4-substituted arylanalogues can be prepared by utilizing the starting materials asoutlined in Scheme 1. The variously substituted aryl chlorides can bereacted with 1,4-diamino or 1-amino-4-nitrobenzene building blocks (with1,2-heteroatoms in the ring) in presence of TEA to give compound 22.

A compound of the present invention or a pharmaceutically acceptablesalt thereof, can be administered as such to a human patient or can beadministered in pharmaceutical compositions in which the foregoingmaterials are mixed with suitable carriers or excipient(s). Techniquesfor formulation and administration of drugs may be found, for example,in REMINGTON'S PHARMACOLOGICAL SCIENCES, Mack Publishing Co., Easton,Pa., latest edition.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or pharmaceutically acceptable salts orprodrugs thereof, with other chemical components, such aspharmaceutically acceptable excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

“Pharmaceutically acceptable excipient” refers to an inert substanceadded to a pharmaceutical composition to further facilitateadministration of a compound. Examples, without limitation, ofexcipients include calcium carbonate, calcium phosphate, various sugarsand types of starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

“Pharmaceutically acceptable salt” refers to those salts which retainthe biological effectiveness and properties of the parent compound. Suchsalts may include: (1) acid addition salt which is obtained by reactionof the free base of the parent compound with inorganic acids such ashydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid,sulfuric acid, and perchloric acid and the like, or with organic acidssuch as acetic acid, oxalic acid, (D)- or (L)-malic acid, maleic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, tartaric acid, citric acid, succinic acid or malonicacid and the like, preferably hydrochloric acid or (L)-malic acid; or(2) salts formed when an acidic proton present in the parent compoundeither is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase such as ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like.

The compound of the present invention may also act, or be designed toact, as a prodrug. A “prodrug” refers to an agent, which is convertedinto the parent drug in vivo. Prodrugs are often useful because, in somesituations, they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent drug is not. The prodrug may also have improved solubility inpharmaceutical compositions over the parent drug. An example, withoutlimitation, of a prodrug would be a compound of the present invention,which is, administered as an ester (the “prodrug”), phosphate, amide,carbamate or urea.

“Therapeutically effective amount” refers to that amount of the compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disorder being treated. In reference to the treatment ofcancer, a therapeutically effective amount refers to that amount whichhas the effect of: (1) reducing the size of the tumor; (2) inhibitingtumor metastasis; (3) inhibiting tumor growth; and/or (4) relieving oneor more symptoms associated with the cancer.

The term “protein kinase-mediated condition” or “disease”, as usedherein, means any disease or other deleterious condition in which aprotein kinase is known to play a role. The term “proteinkinase-mediated condition” or “disease” also means those diseases orconditions that are alleviated by treatment with a protein kinaseinhibitor. Such conditions include, without limitation, cancer and otherhyperproliferative disorders. In certain embodiments, the cancer is acancer of colon, breast, stomach, prostate, pancreas, or ovarian tissue.

The term “Aurora-2 kinase-mediated condition” or “disease”, as usedherein, means any disease or other deleterious condition in which Aurorais known to play a role. The term “Aurora-2 kinase-mediated condition”or “disease” also means those diseases or conditions that are alleviatedby treatment with an Aurora-2 inhibitor.

The term “c-kit-mediated condition” or “disease”, as used herein, meansany disease or other deleterious condition in which c-kit is known toplay a role. The term “c-kit-mediated condition” or “disease” also meansthose diseases or conditions that are alleviated by treatment with ac-kit inhibitor. Such conditions include, without limitation, cancer.

The term “PDGFR-a-mediated condition” or “disease”, as used herein,means any disease or other deleterious condition in which PDGFR-a isknown to play a role. The term “PDGFR-a-mediated condition” or “disease”also means those diseases or conditions that are alleviated by treatmentwith a PDGFR-a inhibitor. Such conditions include, without limitation,cancer.

As used herein, “administer” or “administration” refers to the deliveryof an inventive compound or of a pharmaceutically acceptable saltthereof or of a pharmaceutical composition containing an inventivecompound or a pharmaceutically acceptable salt thereof of this inventionto an organism for the purpose of prevention or treatment of a proteinkinase-related disorder.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intravitreal, intraperitoneal, intranasal, or intraocularinjections. In certain embodiments, the preferred routes ofadministration are oral and intravenous.

Alternatively, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.In this way, the liposomes may be targeted to and taken up selectivelyby the tumor.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in any conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, lozenges, dragees, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient. Pharmaceutical preparations for oral use can be made using asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding other suitableauxiliaries if desired, to obtain tablets or dragee cores. Usefulexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol, cellulose preparations such as,for example, maize starch, wheat starch, rice starch and potato starchand other materials such as gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginicacid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. Stabilizers may be added in these formulations, also.Pharmaceutical compositions which may also be used include hard gelatincapsules. The capsules or pills may be packaged into brown glass orplastic bottles to protect the active compound from light. Thecontainers containing the active compound capsule formulation arepreferably stored at controlled room temperature (15-30° C.).

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray using a pressurized pack or a nebulizer and a suitablepropellant, e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds may also be formulated for parenteral administration,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating materials such assuspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of a water soluble form, such as, without limitation,a salt, of the active compound. Additionally, suspensions of the activecompounds may be prepared in a lipophilic vehicle. Suitable lipophilicvehicles include fatty oils such as sesame oil, synthetic fatty acidesters such as ethyl oleate and triglycerides, or materials such asliposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers and/or agents that increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as depot preparations. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. A compound of thisinvention may be formulated for this route of administration withsuitable polymeric or hydrophobic materials (for instance, in anemulsion with a pharmacologically acceptable oil), with ion exchangeresins, or as a sparingly soluble derivative such as, withoutlimitation, a sparingly soluble salt.

A non-limiting example of a pharmaceutical carrier for the hydrophobiccompounds of the invention is a cosolvent system comprising benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer and anaqueous phase such as the VPD cosolvent system. VPD is a solution of 3%w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD cosolvent system (VPD:D5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This cosolvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of such a cosolventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the cosolventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80, the fraction size ofpolyethylene glycol may be varied, other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone, and othersugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Inaddition, certain organic solvents such as dimethylsulfoxide also may beemployed, although often at the cost of greater toxicity.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the protein kinase-modulating compounds of the invention may beprovided as physiologically acceptable salts wherein the claimedcompound may form the negatively or the positively charged species.Examples of salts in which the compound forms the positively chargedmoiety include, without limitation, quaternary ammonium (definedelsewhere herein), salts such as the hydrochloride, sulfate, carbonate,lactate, tartrate, malate, maleate, succinate wherein the nitrogen atomof the quaternary ammonium group is a nitrogen of the selected compoundof this invention which has reacted with the appropriate acid. Salts inwhich a compound of this invention forms the negatively charged speciesinclude, without limitation, the sodium, potassium, calcium andmagnesium salts formed by the reaction of a carboxylic acid group in thecompound with an appropriate base (e.g. sodium hydroxide (NaOH),potassium hydroxide (KOH), calcium hydroxide (Ca(OH)₂), etc.).

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose, e.g., the modulationof protein kinase activity and/or the treatment or prevention of aprotein kinase-related disorder.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. Then, the dosage can be formulated for use inanimal models so as to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture (i.e., the concentrationof the test compound which achieves a half-maximal inhibition of theprotein kinase activity). Such information can then be used to moreaccurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (bothof which are discussed elsewhere herein) for a subject compound. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See, e.g., GOODMAN & GILMAN'S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, Ch. 3, 9^(th) ed., Ed. by Hardman, J., andLimbard, L., McGraw-Hill, New York City, 1996, p. 46.)

Dosage amount and interval may be adjusted individually to provideplasma levels of the active species which are sufficient to maintain thekinase modulating effects. These plasma levels are referred to asminimal effective concentrations (MECs). The MEC will vary for eachcompound but can be estimated from in vitro data, e.g., theconcentration necessary to achieve 50-90% inhibition of a kinase may beascertained using the assays described herein. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

At present, the therapeutically effective amounts of compounds of thepresent invention may range from approximately 2.5 mg/m² to 1500 mg/m²per day. Additional illustrative amounts range from 0.2-1000 mg/qid,2-500 mg/qid, and 20-250 mg/qid.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration, and other procedures known in the art may be employed todetermine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, etc.

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor of human or veterinary administration. Such notice, for example, maybe of the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition. Suitableconditions indicated on the label may include treatment of a tumor,inhibition of angiogenesis, treatment of fibrosis, diabetes, and thelike.

As mentioned above, the compounds and compositions of the invention willfind utility in a broad range of diseases and conditions mediated byprotein kinases, including diseases and conditions mediated by aurora-2kinase, c-kit and/or PDGFR-a. Such diseases may include by way ofexample and not limitation, cancers such as lung cancer, NSCLC (nonsmall cell lung cancer), oat-cell cancer, bone cancer, pancreaticcancer, skin cancer, dermatofibrosarcoma protuberans, cancer of the headand neck, cutaneous or intraocular melanoma, uterine cancer, ovariancancer, colo-rectal cancer, cancer of the anal region, stomach cancer,colon cancer, breast cancer, gynecologic tumors (e.g., uterine sarcomas,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina or carcinoma of thevulva), Hodgkin's Disease, hepatocellular cancer, cancer of theesophagus, cancer of the small intestine, cancer of the endocrine system(e.g., cancer of the thyroid, pancreas, parathyroid or adrenal glands),sarcomas of soft tissues, cancer of the urethra, cancer of the penis,prostate cancer (particularly hormone-refractory), chronic or acuteleukemia, solid tumors of childhood, hypereosinophilia, lymphocyticlymphomas, cancer of the bladder, cancer of the kidney or ureter (e.g.,renal cell carcinoma, carcinoma of the renal pelvis), pediatricmalignancy, neoplasms of the central nervous system (e.g., primary CNSlymphoma, spinal axis tumors, medulloblastoma, brain stem gliomas orpituitary adenomas), Barrett's esophagus (pre-malignant syndrome),neoplastic cutaneous disease, psoriasis, mycoses fungoides, and benignprostatic hypertrophy, diabetes related diseases such as diabeticretinopathy, retinal ischemia, and retinal neovascularization, hepaticcirrhosis, angiogenesis, cardiovascular disease such as atherosclerosis,immunological disease such as autoimmune disease and renal disease.

The inventive compound can be used in combination with one or more otherchemotherapeutic agents. The dosage of the inventive compounds may beadjusted for any drug-drug reaction. In one embodiment, thechemotherapeutic agent is selected from the group consisting of mitoticinhibitors, alkylating agents, anti-metabolites, cell cycle inhibitors,enzymes, topoisomerase inhibitors such as CAMPTOSAR (irinotecan),biological response modifiers, anti-hormones, antiangiogenic agents suchas MMP-2, MMP-9 and COX-2 inhibitors, anti-androgens, platinumcoordination complexes (cisplatin, etc.), substituted ureas such ashydroxyurea; methylhydrazine derivatives, e.g., procarbazine;adrenocortical suppressants, e.g., mitotane, aminoglutethimide, hormoneand hormone antagonists such as the adrenocorticosteriods (e.g.,prednisone), progestins (e.g., hydroxyprogesterone caproate), estrogens(e.g., diethylstilbesterol), antiestrogens such as tamoxifen, androgens,e.g., testosterone propionate, and aromatase inhibitors, such asanastrozole, and AROMASIN (exemestane).

Examples of alkylating agents that the above method can be carried outin combination with include, without limitation, fluorouracil (5-FU)alone or in further combination with leukovorin; other pyrimidineanalogs such as UFT, capecitabine, gemcitabine and cytarabine, the alkylsulfonates, e.g., busulfan (used in the treatment of chronicgranulocytic leukemia), improsulfan and piposulfan; aziridines, e.g.,benzodepa, carboquone, meturedepa and uredepa; ethyleneimines andmethylmelamines, e.g., altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylolmelamine; and the nitrogen mustards, e.g., chlorambucil (usedin the treatment of chronic lymphocytic leukemia, primarymacroglobulinemia and non-Hodgkin's lymphoma), cyclophosphamide (used inthe treatment of Hodgkin's disease, multiple myeloma, neuroblastoma,breast cancer, ovarian cancer, lung cancer, Wilm's tumor andrhabdomyosarcoma), estramustine, ifosfamide, novembrichin, prednimustineand uracil mustard (used in the treatment of primary thrombocytosis,non-Hodgkin's lymphoma, Hodgkin's disease and ovarian cancer); andtriazines, e.g., dacarbazine (used in the treatment of soft tissuesarcoma).

Examples of antimetabolite chemotherapeutic agents that the above methodcan be carried out in combination with include, without limitation,folic acid analogs, e.g., methotrexate (used in the treatment of acutelymphocytic leukemia, choriocarcinoma, mycosis fungiodes, breast cancer,head and neck cancer and osteogenic sarcoma) and pteropterin; and thepurine analogs such as mercaptopurine and thioguanine which find use inthe treatment of acute granulocytic, acute lymphocytic and chronicgranulocytic leukemias.

Examples of natural product-based chemotherapeutic agents that the abovemethod can be carried out in combination with include, withoutlimitation, the vinca alkaloids, e.g., vinblastine (used in thetreatment of breast and testicular cancer), vincristine and vindesine;the epipodophyllotoxins, e.g., etoposide and teniposide, both of whichare useful in the treatment of testicular cancer and Kaposi's sarcoma;the antibiotic chemotherapeutic agents, e.g., daunorubicin, doxorubicin,epirubicin, mitomycin (used to treat stomach, cervix, colon, breast,bladder and pancreatic cancer), dactinomycin, temozolomide, plicamycin,bleomycin (used in the treatment of skin, esophagus and genitourinarytract cancer); and the enzymatic chemotherapeutic agents such asL-asparaginase.

Examples of useful COX-II inhibitors include Vioxx, CELEBREX(celecoxib), valdecoxib, paracoxib, rofecoxib, and Cox 189.

Examples of useful matrix metalloproteinase inhibitors are described inWO 96/33172 (published Oct. 24, 1996), WO 96/27583 (published Mar. 7,1996), European Patent Application No. 97304971.1 (filed Jul. 8, 1997),European Patent Application No. 99308617.2 (filed Oct. 29, 1999), WO98/07697 (published Feb. 26, 1998), WO 98/03516 (published Jan. 29,1998), WO 98/34918 (published Aug. 13, 1998), WO 98/34915 (publishedAug. 13, 1998), WO 98/33768 (published Aug. 6, 1998), WO 98/30566(published Jul. 16, 1998), European Patent Publication 606,046(published Jul. 13, 1994), European Patent Publication 931,788(published Jul. 28, 1999), WO 90/05719 (published May 31, 1990), WO99/52910 (published Oct. 21, 1999), WO 99/52889 (published Oct. 21,1999), WO 99/29667 (published Jun. 17, 1999), PCT InternationalApplication No. PCT/IB98/01113 (filed Jul. 21, 1998), European PatentApplication No. 99302232.1 (filed Mar. 25, 1999), Great Britain patentapplication number 9912961.1 (filed Jun. 3, 1999), U.S. Pat. No.5,863,949 (issued Jan. 26, 1999), U.S. Pat. No. 5,861,510 (issued Jan.19, 1999), and European Patent Publication 780,386 (published Jun. 25,1997), all of which are incorporated herein in their entireties byreference. Preferred MMP-2 and MMP-9 inhibitors are those that havelittle or no activity inhibiting MMP-1. More preferred are those thatselectively inhibit MMP-2 and/or MMP-9 relative to the othermatrix-metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).

Some specific examples of MMP inhibitors useful in the present inventionare AG-3340, RO 32-3555, RS 13-0830, and compounds selected from:3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methylbenzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[(4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino]-propionicacid;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionicacid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts and solvates ofthese compounds.

Other anti-angiogenesis agents, other COX-II inhibitors and other MMPinhibitors, can also be used in the present invention.

An inventive compound can also be used with other signal transductioninhibitors, such as agents that can inhibit EGFR (epidermal growthfactor receptor) responses, such as EGFR antibodies, EGF antibodies, andmolecules that are EGFR inhibitors; VEGF (vascular endothelial growthfactor) inhibitors; and erbB2 receptor inhibitors, such as organicmolecules or antibodies that bind to the erbB2 receptor, such asHERCEPTIN (Genentech, Inc., South San Francisco, Calif.). EGFRinhibitors are described in, for example in WO 95/19970 (published Jul.27, 1995), WO 98/14451 (published Apr. 9, 1998), WO 98/02434 (publishedJan. 22, 1998), and U.S. Pat. No. 5,747,498 (issued May 5, 1998), andsuch substances can be used in the present invention as describedherein.

EGFR-inhibiting agents include, but are not limited to, the monoclonalantibodies C225 and anti-EGFR 22Mab (ImClone Systems, Inc., New York,N.Y.), the compounds ZD-1839 (AstraZeneca), BIBX-1382 (BoehringerIngelheim), MDX-447 (Medarex Inc., Annandale, N.J.), and OLX-103 (Merck& Co., Whitehouse Station, N.J.), and EGF fusion toxin (Seragen Inc.,Hopkinton, Mass.).

These and other EGFR-inhibiting agents can be used in the presentinvention. VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.,South San Francisco, Calif.), can also be combined with an inventivecompound. VEGF inhibitors are described in, for example, WO 01/60814 A3(published Aug. 23, 2001), WO 99/24440 (published May 20, 1999), PCTInternational Application PCT/IB99/00797 (filed May 3, 1999), WO95/21613 (published Aug. 17, 1995), WO 99/61422 (published Dec. 2,1999), U.S. Pat. No. 5,834,504 (issued Nov. 10, 1998), WO 01/60814, WO98/50356 (published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar.16, 1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No.5,792,783 (issued Aug. 11, 1998), WO 99/10349 (published Mar. 4, 1999),WO 97/32856 (published Sep. 12, 1997), WO 97/22596 (published Jun. 26,1997), WO 98/54093 (published Dec. 3, 1998), WO 98/02438 (published Jan.22, 1998), WO 99/16755 (published Apr. 8, 1999), and WO 98/02437(published Jan. 22, 1998), all of which are incorporated herein in theirentireties by reference. Other examples of some specific VEGF inhibitorsuseful in the present invention are IM862 (Cytran Inc., Kirkland,Wash.); anti-VEGF monoclonal antibody of Genentech, Inc.; and angiozyme,a synthetic ribozyme from Ribozyme (Boulder, Colo.) and Chiron(Emeryville, Calif.). These and other VEGF inhibitors can be used in thepresent invention as described herein. pErbB2 receptor inhibitors, suchas GW-282974 (Glaxo Wellcome plc), and the monoclonal antibodies AR-209(Aronex Pharmaceuticals Inc., The Woodlands, Tex.) and 2B-1 (Chiron),can furthermore be combined with an inventive compound, for example,those indicated in WO 98/02434 (published Jan. 22, 1998), WO 99/35146(published Jul. 15, 1999), WO 99/35132 (published Jul. 15, 1999), WO98/02437 (published Jan. 22, 1998), WO 97/13760 (published Apr. 17,1997), WO 95/19970 (published Jul. 27, 1995), U.S. Pat. No. 5,587,458(issued Dec. 24, 1996), and U.S. Pat. No. 5,877,305 (issued Mar. 2,1999), which are all hereby incorporated herein in their entireties byreference. ErbB2 receptor inhibitors useful in the present invention arealso described in U.S. Pat. No. 6,284,764 (issued Sep. 4, 2001),incorporated in its entirety herein by reference. The erbB2 receptorinhibitor compounds and substance described in the aforementioned PCTapplications, U.S. patents, and U.S. provisional applications, as wellas other compounds and substances that inhibit the erbB2 receptor, canbe used with an inventive compound, in accordance with the presentinvention.

An inventive compound can also be used with other agents useful intreating cancer, including, but not limited to, agents capable ofenhancing antitumor immune responses, such as CTLA4 (cytotoxiclymphocite antigen 4) antibodies, and other agents capable of blockingCTLA4; and anti-proliferative agents such as other farnesyl proteintransferase inhibitors, for example the farnesyl protein transferaseinhibitors described in the references cited in the “Background”section, of U.S. Pat. No. 6,258,824 B1.

The above method can be also be carried out in combination withradiation therapy, wherein the amount of an inventive compound incombination with the radiation therapy is effective in treating theabove diseases.

Techniques for administering radiation therapy are known in the art, andthese techniques can be used in the combination therapy describedherein. The administration of the compound of the invention in thiscombination therapy can be determined as described herein.

The invention will be better understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES

A structure-based design approach was used employing three-dimensionalstructural modeling of protein kinase catalytic sites and their bindingrelationship with inhibitor compounds to design the inventive compoundsdescribed herein. Homology modeling of protein kinases has been used topredict and analyze the three-dimensional structures of these proteins.A suite of programs that employs PSI-BLAST (NCBI), THREADER (HGMPResource Center, Hinxton, Cambs, CB10 1SA, UK), 3D-PSSM(three-dimensional position scoring matrix) (HGMP) and SAP programs wasused to determine the optimal template for homology modeling of aurora-1and aurora-2 kinases, c-kit tyrosine kinase receptor and PDGFR-A. Thecrystal structure of the activated form of bovine cAMP-dependent proteinkinase was identified as the best template and subsequently used foraurora kinase homology modeling using molecular dynamics (MD)simulations in INSIGHT II (version 2000, Accelrys Inc.) running on anIndigo2 workstation (Silicon Graphics, Inc.). The modeled aurora-2structure was docked with known S/T kinase and aurora-2 kinaseinhibitors using the binary complex of cAMP-dependent PK-Mn²⁺-adenylylimidodiphosphate (AMP-PNP). The calculated binding energies from thedocking analysis are in agreement with experimental IC₅₀ values obtainedfrom an in vitro kinase assay, which uses histone H1 or myelin basicprotein (MBP) phosphorylation to assess inhibitory activity. Theaurora-2 structural model provided a rational basis for site-directedmutagenesis studies of the active site and in silico screening ofchemical databases, thereby allowing the design of novel aurora-2 kinaseinhibitors described herein, e.g., pyrimido[4,5-b]indoles,benzothieno[3,2-d]pyrimidones, benzofuranopyrimidines and6,7-quinazolines.

The crystal structures of the activated forms of VEGFR2 and FGFR₁protein kinase receptors were identified as the best templates andsubsequently used for c-kit homology modeling using molecular dynamics(MD) simulations in INSIGHT II (version 2000, Accelrys Inc.) running onan Indigo2 workstation (Silicon Graphics, Inc.). Then the modeled c-kitbinding site structure was docked with known c-kit inhibitors (STI571,CT52923, PD173955 and SU5614).

The c-kit structural model provided a basis for electronically mutatingthe active site and using another computer program to screen chemicaldatabases, thereby allowing the design of novel c-kit kinase inhibitors.For example, on the basis of docking chemicals in the active site, itwas determined that certain compound classes(4-piprazinylpyrimido[4,5-b]indoles, benzothieno[3,2-d],benzofuranopyrimidines and quinazolines containing analogs, see FIG. 12)could replace the 6,7-dimethoxy quinazoline and the adenine base of ATP,thereby allowing new hydrogen bonding and hydrophobic interactionswithin the ATP binding pocket.

Example 1 Aurora Sequence and Structure Analysis

A PSI-BLAST search (NCBI) was performed with the sequence of the kinaseportion of human aurora-1 and aurora-2 kinases and high sequencesimilarities were found to porcine heart bovine cAMP-dependent kinase(PDB code 1CDK), murine cAMP-dependent kinase (1APM), and C. eleganstwitchin kinase (1KOA), whose three-dimensional structures have beensolved. The three manually aligned S/T kinase domain sequences withtheir respective secondary structures were viewed in Clustal X (FIG. 2).

The aurora-1 or aurora-2 sequences were inputted into the tertiarystructure prediction programs THREADER and 3D-PSSM, which compareprimary sequences with all of the known three-dimensional structures inthe Brookhaven Protein Data Bank. The output is composed of theoptimally aligned, lowest-energy, three-dimensional structures that aresimilar to the aurora kinases. The top structural matches were bovine1CDK, murine 1APM and 1KOA, confirming that the aurora kinase proteinsare structurally conserved.

Example 2 Aurora Homology Modeling

The 1CDK, 1APM and 1KOA tertiary structures provided thethree-dimensional templates for the homology modeling of aurora-1 andaurora-2 kinases. The crystal structure coordinates for the aboveserine/tyrosine kinase domains were obtained from the Protein Data Bank.These domains were pair-wise superimposed onto each other using theprogram SAP. The structural alignments produced by the SAP program werefine-tuned manually to better match residues within the regularsecondary structural elements.

Structural models were built of aurora-1 and 2 using 1CDK as thetemplate structure. The final aurora-2 model (FIG. 3) was analyzed usingProfile-3D. The Profile-3D and 3D-1D score plots of the model werepositive over the entire length of protein in a moving-window scan tothe template structure. Additionally, the PROCHECK program was used toverify the correct geometry of the dihedral angles and the handedness ofthe aurora-2 model.

Example 3 Aurora Molecular Dynamics (MD) and Docking Analysis

MD simulations were performed in the canonical ensemble (NVT) at 300° K.using the CFF force field implemented in the Discover_(—)3 program(version 2.9.5). Dynamics were equilibrated for 10 picoseconds with timesteps of 1 femtosecond and continued for 10-picosecond simulations. Anonbonded cutoff distance of 8 Å and a distance-dependent dielectricconstant (∈=5rij) for water were used to simulate the aqueous media. Allof the bonds to hydrogen were constrained. Dynamic trajectories wererecorded every 0.5 picoseconds for analysis. The resulting low energystructure was extracted and energy-minimized to 0.001 kcal/mol/Å. Toexamine the conformational changes that occur during MD, the root meansquare (rms) deviations were calculated from trajectories at0.5-picosecond intervals and compared to the Cα backbone ofcAMP-dependent PK. The rms deviation for the two superimposed structureswas 0.42 Å. Furthermore, the rms deviations were calculated for theprotein backbone (0.37 Å) and the active-site pocket (0.41 Å) and werecompared with crystal structure before the docking experiments. Theresulting aurora-2 structure served as the starting model for dockingstudies.

For docking analysis, the ligand structures were obtained from fivecrystal structure complexes of cAMP-dependent PK bound with AMP-PNP,staurosporine, H-89, H-7, or H-8 and from structures that wereempirically built and energy minimized (KN-93, ML-7, and6,7-dimethoxyquinazoline) (FIG. 4) in the INSIGHT II program. The heavyatoms from AMP-PNP were used as sphere centers for the dockingprocedures. Docking simulations were performed at 500° K. with 100femtosecond/stage (total of 50 stages), quenching the system to a finaltemperature of 300° K. The whole complex structure was energy minimizedusing 1000 steps. This provided 10 structures from the simulatedannealing (SA) docking, and their generated conformers were clusteredaccording to rms deviation. The lowest global structure complexes wereused to calculate intermolecular binding energies.

Example 4 Design Strategy for Aurora-2 Kinase Inhibitors

Based on the binding mode of several competitive inhibitors of aurora-2kinase depicted in FIG. 5, we explored the structural moieties requiredfor aurora-2 kinase inhibition. The structures are shown superimposed.The enzyme active site has been clipped. We evaluated the functionalrelationship among the known serine/threonine kinase inhibitors bystructure-based design and molecular modeling approaches. In aurora-2kinase, the NH and C═O groups in Glu211 and Ala213 and the Gly-richpocket residues appear to be most important in inhibitor binding. Thesestructures are hydrogen-bond donors/acceptors and are in all reportedS/T kinase structures. Residues Asp274 and Lys141 are also veryimportant in hydrogen bonding. Additionally, our modeling indicated thatthe flat aromatic rings of the aurora-2 inhibitors occupy the ATPbinding pocket around Glu221 and are surrounded by residues Val147 andAla213. Also, structural alignments of known S/T kinase inhibitors showtwo shared structural motifs with similarly placed nitrogens andsix-membered aromatic rings, suggesting that these compounds havesimilar binding patterns.

To identify new chemicals that satisfy these structural requirements, ade novo design approach was employed using the graphical chemicalmodeling program LUDI (Accelrys). Initially, lead structures (purinebase, quinazoline, isoquinazoline and indole rings) were dissected intocore templates and two additional fragments (FIG. 6), which formed thebasis of a built-in compound library. Then template structures wereobtained from the Available Chemical Directory (ACD). The compounds withmolecular weight >350 were selected, and chemical skeletons orfunctional groups that were unacceptable for the development of leadcompounds were omitted from the library. An in-house compound librarycontaining the identified templates was built and utilized in LUDIsearch procedures. Additionally, three tricyclic quinazoline typetemplates were identified apart from the isoquinolines and quinazolines.From the LUDI fragment library, structurally similar fragments wereobtained for fragments 1 and 2 (FIG. 6). Fragment selection was based onthe following criteria: (1) molecular weight <350, (2) at least twohydrogen bond donor/acceptor groups, (3) at least three rings, and (4)correct position and orientation with respect to lead compounds withinthe ATP binding pocket. The template and fragments were linked in LUDIlink mode to confirm their binding mode for the newly built structures.Several combinations of structures were designed by keeping the requiredpharmacophores identified from ACD and LUDI fragment searches. More than90 compounds were built using this structure-based scaffold approach.Further, these compounds were screened to exclude molecules that werenot complementary to the ATP binding pocket by the FlexX docking method(Tripos, St. Louis, Mo.). Forty-two compounds (FIGS. 7A-7D) were foundto have the optimal number of H-bonds, position and orientation withinthe ATP binding pocket and FlexX scoring.

Example 5 Chemical Synthesis of Kinase Inhibitors

General Methods. ¹HNMR was run on a Unity 300-MHz NMR Spectrophotometer(Varian, Palo Alto, Calif.). The chemical shifts are relative to thetrace proton signals of the deuterated solvent. Coupling constants, J,are reported in Hz and refer to apparent peak multiplicity rather thancoupling constants. Fast atom bombardment (FAB) measurements have beencarried out on a mass spectrometer HX-110 instrument (JEOL, Akishima,Japan) equipped with a conventional Xe gun. A mixture matrix ofglycerol:thioglycerol:mNBA (meta-nitrobenzyl alcohol) 50:25:25containing 0.1% of trifluoroacetic acid (TFA) was used as the matrix forfast atom bombardment (FAB). For accurate mass measurements,polyethylene glycol (PEG) was used as the internal standard. Flashcolumn chromatography was performed on silica gel 60, purchased fromSpectrum. Combustion analysis (CHNS) was performed by Desert AnalyticsLaboratory, Tucson, Ariz. Synthesis of4-chloro-6,7-dimethoxyquinazoline,4-chloro-benzothieno[3,2-d]pyrimidone, 4-chloro-benzofuranopyrimidoneand 4-chloropyrimido[4,5-b]indole is carried out by reaction withvarious dihydro-quinazolines using formamide HCl/formaide at 180-190° C.followed by the addition of Vilsmeier's reagent to obtain4-chloro-quinazolines. General methods for synthesizing these buildingblocks are illustrated in FIG. 8.

The 4-chloro-quinazoline building blocks are reacted with2-amino-5-nitropyrimidines, and various unsubstituted o-, m- orp-6-membered aromatic rings, or containing a direct bond, NHCO, NHCSNH,SO₂NH, NHSO₂, NHCH₂Ph, aminopyrazoles, amino-substituted oxadiazoles,thiadiazoles or triazoles, to give the 4-substituted tricyclic andquinazoline series of compounds (e.g., FIG. 8).

The synthesis of the thiourea-containing compounds was carried out usingthe following general procedure. Piprenolamine, sulfadiazine and/orsubstituted aromatic amines were slowly added to a solution ofthiophosgene in dichloromethane, followed by the addition oftriethylamine on an ice bath. After the reaction mixture was stirred for4 hours, 4-chloro-quinazolines or tricyclic building blocks were addedand the resulting mixture was stirred overnight at room temperature.Methanol was added to quench the excess thiophosgene, and the residuewas purified by silica gel column chromatography after removal ofsolvent.

Example 6 4-chloro-tricyclic and quinazoline Building Blocks

The 4-chloro-tricyclic and quinazoline building blocks were synthesizedusing literature methods (Pandey, A., et al., J. Med. Chem. 2002,45:3772-93; Matsuno, K., et al., J. Med. Chem. 2002, 45:3057-66;Matsuno, K., et al., J. Med. Chem. 2002, 45:4513-23; and Venugopalan,B., et al., J. Heterocycl. Chem. 1988, 25:1633-39). As shown in FIG. 9,these were converted to the corresponding 4-piperazine derivatives byrefluxing with piperazine in pyridine or dioxane.

Example 7 N-Pyrimidin-2-yl-4-thioformylamino-benzenesulfonamide chloride(1d)

To a stirred solution of sulfadiazine (192 mg, 0.77 mmol) indichloromethane (20 mL) were slowly added thiophosgene (0.06 mL, 0.83mmol) and triethylamine (0.05 mL, 0.32 mmol) under cooling with an icebath. After the reaction mixture was stirred for 5 hours at roomtemperature, it was washed with water and brine, dried over anhydroussodium sulfate, filtered, evaporated and dried under vacuum; and theproduct was used immediately for the next reaction.

Example 8 4-(6,7-Dimethoxy-quinazolin-4-yl)-piperazine-1-carbothioicacid [4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide (HPK16)

To a solution of 4-(1-piperazinyl)-6,7-dimethoxy quinazoline (200 mg,0.73 mmol) and pyridine (0.5 mL, 6.4 mmol) in dichloromethane (20 mL)was added a solution of product 1d in dichloromethane (20 mL) andstirred overnight. Methanol was added for quenching excess thiophosgene,and the residue after removal of solvent was purified by silica gelcolumn chromatography eluting with 5% methanol/dichloromethane andfurther recrystallized from dichloromethane/hexane to give 80 mg (20%).

¹H NMR (CDCl₃, 300 MHZ) δ 3.85(s, 4H), 3.98(s, 3H), 4.02(s, 3H), 4.11(s,4H), 6.98(m, 1H), 7.08(s, 1H), 7.32(d, 2H), 7.88(s, 1H), 8.00(d, J=6.7Hz, 2H), 8.62(d, 2H), 8.66(s, 1H). FAB HRMS [M+H]⁺ calcd forC₂₅H₂₆N₈O₄S₂: 566.1518; found 567.1597. Combustion Analysis:C₂₅H₂₆N₈O₄S₂ Requires C, 52.99%; H, 4.62%; N, 19.77%; O, 11.29%; S,11.32%. Found C, 53.27%; H, 4.94%; N, 19.99%; O, 11.57%; S, 11.64%.

Example 94-(6,7-Dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioicacid [4-(Pyrimidin-2-ylsulfamoyl)-phenyl]-amide (HPK62/MP-235)

To a solution of 6,7-dimethoxy-4-piperazino-9H-pyrimido[4,5-b]indole(200 mg, 0.64 mmol) and pyridine (0.5 mL, 6.4 mmol) in dichloromethane(20 mL) was added a solution of product 1d in dichloromethane (20 mL)and the mixture was stirred overnight. Methanol was added to quenchexcess thiophosgene, and the residue after removal of solvent waspurified by silica gel column chromatography, eluting with 5%methanol/dichloromethane and was further recrystallized fromdichloromethane/hexane to give 50 mg (16%).

¹HNMR (DMSO-d₆, 300 MHZ) δ 3.75(s, 4H), 3.87(s, 3H), 3.88(s, 3H),4.19(s, 4H), 7.04-7.06 (m, 1H), 7.07(s, 1H), 7.24(s, 1H), 7.53(d, J=8.4Hz, 2H), 7.90(d, J=8.4 Hz, 2H), 8.44(s, 1H), 8.51(d, J=4.8 HZ, 2H),9.72(s, 1H, —NH), 12.01 (s, 1H, —NH). FAB HRMS [M+H]⁺ calcd forC₂₇H₂₇N₉O₄S₂: 605.1627; found 606.1699. Combustion Analysis: RequiresC₂₇H₂₇N₉O₄S₂ Requires C, 53.54%; H, 4.49%; N, 20.81%; O, 10.57%; S,10.59%. Found C, 53.84%; H, 4.91%; N, 21.21%; O, 11.87%; S, 8.17%.

Example 10 Aurora-2 Kinase Inhibition Assay

In this assay kinase activity is determined by quantifying the amount ofATP remaining in solution following the kinase reaction by measuring thelight units (LU) produced by luciferase using a luminometer. Percentinhibition was determined for individual compounds by comparingluminometer readings of drug-treated reactions to controls containing nodrug (DMSO control) and no Aurora-2 enzyme (ATP control) in thefollowing equation:

${{Percent}\mspace{14mu}{I{nhibition}}} = {\frac{{LU}_{drug} - {LU}_{DMSO}}{{LU}_{ATP} - {LU}_{DMSO}} \times 100}$

In a 50 μl reaction, recombinant aurora-2 kinase produced in sf9 cells(Imgenex, San Diego, Calif.) was incubated at 30° C. for two hours with62.5 μM Kemptide (Calbiochem, San Diego, Calif.), 3 μM ATP (Invitrogen,Carlsbad, Calif.) and kinase reaction buffer (40 mM Tris-HCl, 10 mMMgCl₂ and 0.1 μg/μl bovine serum albumin (BSA)). This reaction wascarried out in the presence of drug substances, which had beenpreviously diluted to desired concentrations in DMSO. After incubation,50 μl of Kinase-Glo® (Promega, Inc., Madison, Wis.) solution was addedto each reaction mixture and allowed to equilibrate for 10 minutes atroom temperature. Kinase-Glo solution contains luciferase enzyme andluciferin, which react with ATP to produce light. Kinase activity isdetermined by quantifying the amount of ATP remaining in solutionfollowing the kinase reaction by measuring the light units (LU) producedby luciferase using a luminometer (PerkinElmer, Boston, Mass.). FIG. 10shows the degree of inhibition of aurora-2 kinase activity byillustrative compounds of the invention, including HPK56 (StructureIII-1-3), HPK61 (Structure II-2-7), HPK60 (Table 4; Structure 34-4),HPK59 (Structure III-1-5), AKS301 (Table 6; Structure 38-16), AKS110(Table 6, Structure 38-14), AKS300 (Table 6, Structure 38-15), AKS302(Table 6, Structure 38-17), HPK16 (Structure IV-1-3) and HPK62(Structure II-2-6), in addition to several precursors and known kinaseinhibitors (e.g., HMN-176, Quincl, trioxd, trithiad, azpyram, Quinam,Suldz, Gmocnhcl and trinhcl). The synthesized compound HPK62 had thehighest inhibition, and compound HPK16 had the second highest inhibitionof the tested compounds.

The drug concentration at which 50% of aurora-2 kinase activity wasinhibited (IC₅₀) was determined for illustrative compounds and theresults shown in FIG. 11. HPK16 (Structure IV-1-3) and HPK62 (StructureII-2-6) were particularly effective inhibitors. A range of chemicaldoses was tested, and graphed, as shown in FIG. 11. The IC₅₀ values forthe compounds are shown below in Table 1.

TABLE 1 Compound Designation Structure IC₅₀ HPK16 IV-1-3 4.7 μM HPK62II-2-6 0.9 μM AKS110 38-14  36 μM

Example 11 c-kit Sequence and Structure Analysis

The known sequence of the c-kit tyrosine kinase active domain was usedin a PSI-BLAST search (NCBI) of non-redundant database of sequences.Top-ranked sequences for which three-dimensional structures of tyrosinekinase (TK) domains also were available were the vascular endothelialgrowth factor receptor (VEGFR2, or 1VR2) and fibroblast growth factorreceptor 1 (FGFr1, or 1FGI). These sequences, along with those ofPDGFR-α, PDGFR-β and c-Abl, were manually aligned by their kinase domainsequences and their respective secondary structures and viewed inClustal X (FIG. 13).

The c-kit TK domain sequence was inputted into the tertiary structureprediction programs THREADER and 3D-PSSM, which compare primarysequences with all of the known three-dimensional structures in theBrookhaven Protein Data Bank. The output was composed of the optimallyaligned, lowest-energy, three-dimensional structures that were similarto c-kit. The top structural matches were VEGFR2 and FGFr1, confirmingthat these proteins are structurally conserved.

Example 12 c-kit Homology Modeling

VEGFR2 and FGFr1 structures provided the three-dimensional templates forthe homology modeling of c-kit. The crystal structure coordinates forthe above TK domains were obtained from the Protein Data Bank. Thesedomains were pair-wise superimposed onto each other using the SAPprogram. The structural alignments from SAP were fine-tuned manually tobetter match residues within the regular secondary structural elements.The modeling software used was Insight II (version 2000, Accelrys Inc.),running on a Silicon Graphics Indigo2 workstation under the Unixoperating system. After the model building processes were complete, aseries of minimizations were performed to relax the structure. The finalc-kit model (FIG. 14) was examined using 3D-profile. Additionally,PROCHECK was used to verify the correct geometry of the dihedral anglesand the handedness of the model-built structure.

Example 13 c-kit Molecular Dynamics (MD) and Docking Analysis

The 3D c-kit model served as the starting point for docking studies ofCT662923 and STI571 (GLEEVEC™). MD simulations were performed in thecanonical ensemble (NVT) at 300° K. using the CFF force fieldimplemented in Discover_(—)3 (version 2.9.5; Accelrys). Dynamics wereequilibrated for 10 picoseconds with time steps of 1 femtosecond andcontinued for 10-picosecond simulations. The nonbonded cutoff distanceof 8 Å and a distance-dependent dielectric constant (∈=5rij) for waterwere used to simulate the aqueous media. All of the bonds to hydrogenwere constrained. Dynamic trajectories were recorded every 0.5picoseconds for analysis. The resulting low energy structure wasextracted and energy-minimized to 0.001 kcal/mol/Å. To examine theconformational changes that occur during MD, the root mean square (rms)deviations were calculated from trajectories at 0.5-picosecond intervalsand compared to the Cα backbones of VDGFR and FDFr TK. The resultingc-kit structure served as the starting model for docking studies.

For docking studies, the starting model structures of ligands were fromthe known c-kit tyrosine kinase inhibitors of CT52923 (FIG. 15A) andSTI571 (GLEEVEC™) (FIG. 15B) and were empirically built and energyminimized. The heavy atoms from FGFr kinase domain were used as spherecenters for the docking procedures. Docking simulations were performedat 500° K. with 100 femtosecond/stage (total of 50 stages), quenchingthe system to a final temperature of 300° K. The whole complex structurewas energy minimized using 1000 steps. This provided 10 structures fromthe simulated annealing (SA) docking, and their generated conformerswere clustered according to rms deviation. The lowest energy globalstructure complexes were used to calculate intermolecular bindingenergies.

Example 14 c-kit FlexX Docking

FlexX docking was performed in the Sybyl 6.8 program (Tripos, St. Louis,Mo.). The structures of ligands used for docking were the crystalstructure of STI571 with the Abl tyrosine kinase and the CT52923 whichwas empirically built and energy-minimized in Insight II. Systematicconformational searches were performed on each of the minimized ligandsusing 10-picosecond MD simulations at 300° K. For docking with CT 52923and STI571, the position of the SU5402, an indolinone analog wasretained from its crystal structure of 1FGI in which the indolinoneserved as a template for field-fit alignments with the quinazoline andpyrimidoindole-containing compounds. The indolinone analog was thenremoved from the field-fit alignment, and each of the other ligands wasdocked into the active site pocket with a similar position andorientation to that of CT52923 (FIG. 15A) and STI571 (FIG. 15B) usingFlexX multiple molecule docking methodology.

Based on our analysis of the binding mode of CT52923 and STI571 depictedin FIGS. 15A and 15B, respectively, the presence of two sharedstructural motifs of similarly placed hydrogen bond acceptors andsix-membered aromatic rings suggested that these compounds may beexhibiting some common binding regions. Based on these two sets ofalignments, a phenylamine-pyrimidine moiety was introduced at position 4of CT52923 and the position of this substitution was furtherrationalized by FlexX docking and molecular dynamics simulation.

Example 15

Design Strategy

To identify new chemicals that satisfy the above-identified structuralrequirements, a de novo design approach was employed using the graphicalchemical modeling program LUDI (Accelrys). Initially, the leadstructures (purine base, phenylamino pyrimidines,pyrimido[4,5-b]indoles, benzofurano and benzothieno[3,2-d]pyrimidenes,pyrido[3,2-dpyrimidenes, quinazolines, and indole rings) were dissectedinto core templates and two additional fragments (FIG. 16), which formedthe basis of our built-in compound library. This built-in library,containing the identified templates, together with the LUDI/ACDdatabases, was used in the search procedures within the Insight IIprogram (Accelrys). In addition to the known quinazoline andphenylamino-pyrimidine moieties, which are the tricyclicpyrimido[4,5-b]indoles, benzofuranopyrimidines, andbenzothieno[3,2-d]pyrimidines (Scheme 1), three novel hits wereidentified from the LUDI search. Further, fragment searches wereperformed for the replacement of the sugar and α-, β-, and γ-phosphatebinding regions (e.g., Mohammedi, M., et al., Science, 1997, 276:955-960). The piperazine, thiourea, and piperonylamine fragments ofCT52923 were bonded in the LUDI link mode at the 4-position of the newtricyclic moieties. The position and orientation of this substitutionwere further rationalized by LUDI FlexX. docking (Tripos, St. Louis,Mo.) within the Sybyl software, and molecular dynamics simulations.Finally, 4-amino-N-(2-pyrimidinyl)benzene sulfonamide (sulphadiazine)fragments were identified from the LUDI/ACD databases. These fragmentswere also linked at the 4-position of the tricyclic structural moieties.The fragment selection was based on hydrogen bond donor/acceptor groupsand correct position and orientation with respect to the lead compounds(FIG. 12) within the ATP binding pocket.

Several combinations of structures were designed by keeping the requiredcore structures identified from ACD and LUDI fragment searches. Morethan 60 compounds were built using this structure-based scaffoldapproach. Further, these compounds were screened to exclude moleculesthat were not complementary to the ATP binding pocket (Leu595, Phe600,Val603, Ala621, Val654, Thr670, Glu671, Tyr672, Cys673, Gly676, Asp677,Asn739, Leu741, and Asp752) by the FlexX docking method. Compounds 1-7of FIG. 17 (HPK61 (II-2-7), HPK62 (II-2-6), HPK56 (III-1-3), HPK59(III-1-5), HPK57 (III-1-4), HPK60 (34-4) and HPK16 (IV-1-3),respectively) were found to have the optimal number of hydrogen bonds,positions and orientations within the ATP binding pocket and the optimalFlexX scoring (kJ/mol). These seven compounds were synthesized andevaluated for c-kit and PDGFR tyrosine kinase inhibitory activity.

Example 16 Chemical Synthesis

General Methods. ¹HNMR was run on a Unity 300-MHz NMR Spectrophotometer(Varian, Palo Alto, Calif.). The chemical shifts are relative to thetrace proton signals of the deuterated solvent. Coupling constants, J,are reported in Hz and refer to apparent peak multiplicity rather thancoupling constants. Fast atom bombardment (FAB) measurements have beencarried out on a mass spectrometer HX-110 instrument (JEOL, Akishima,Japan) equipped with a conventional Xe gun. A mixture matrix ofglycerol:thioglycerol:mNBA (meta-nitrobenzyl alcohol) 50:25:25containing 0.1% of trifluoroacetic acid (TFA) was used as the fast atombombardment (FAB) matrix. For accurate mass measurements, polyethyleneglycol (PEG) was used as the internal standard. Flash columnchromatography was performed on silica gel 60, purchased from Spectrum.Combustion analysis (CHNS) was performed by Desert Analytics Laboratory,Tucson, Ariz.

The synthesis of 4-piperazinylpyrimido[4,5-b]indoles (1b),benzofuranopyrimidines (2b), benzothieno[3,2-d]pyrimidines (3b), andquinazoline (4b) derivatives is depicted in FIG. 20. 4-Chloro-tricyclicand quinazoline building blocks (1a-4a) were synthesized usingliterature methods. (Pandey, A., et al., J. Med. Chem. 2002, 45:3772-93;Matsuno, K., et al., J. Med. Chem. 2002, 45:3057-66; Matsuno, K., etal., J. Med. Chem. 2002, 45:4513-23; and Venugopalan, B., et al., J.Heterocycl. Chem. 1988, 25:1633-39.) These were converted to thecorresponding 4-piperazine derivatives by refluxing with piperazine inpyridine or dioxane. Piperonylamine or sulfadiazine were slowly added toa solution of thiophosgene in dichloromethane while cooling with an icebath. The resulting mixture was stirred for four hours at roomtemperature, which gave 1c or 1d, as shown in FIG. 21. Compounds 1c or1d were further reacted with 4-piperazine-substituted tricyclic orquinazoline derivatives in dichloromethane and stirred overnight at roomtemperature. To quench excess isothiocyanate, methanol was added, andafter removal of solvent, the residue was purified by silica gelchromatography to give compounds 1-7 of FIG. 17 in approximately 20-40%yields.

Example 17 N-Benzo[1,3]dioxol-5-ylmethyl-thioformamide chloride (1c)

To a stirred solution of piperonylamine (0.1 mL, 0.77 mmol) indichloromethane (20 mL) was slowly added thiophosgene (0.06 mL, 0.83mmol) under cooling with an ice bath. After the reaction mixture wasstirred for four hours at room temperature, it was washed with water andbrine, dried over anhydrous sodium sulfate, filtered, evaporated anddried under vacuum; and the product was used immediately for the nextreaction.

Example 18 N-Pyrimidin-2-yl-4-thioformylamino-benzenesulfonamidechloride (1d)

To a stirred solution of sulfadiazine (192 mg, 0.77 mmol) indichloromethane (20 mL) were slowly added thiophosgene (0.06 mL, 0.83mmol) and triethylamine (0.05 mL, 0.32 mmol) under cooling with an icebath. After the reaction mixture was stirred for five hours at roomtemperature, it was washed with water and brine, dried over anhydroussodium sulfate, filtered, evaporated and dried under vacuum. The productwas used immediately for the next reaction.

Example 194-(6,7-Dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioicacid (benzo[1,3]dioxol-5-ylmethyl)-amide (1)

To a solution of 6,7-dimethoxy-4-piperazino-9H-pyrimido[4,5-b]indole(200 mg, 0.64 mmol) and pyridine (0.5 mL, 6.4 mmol) in dichloromethane(20 mL) was added the solution of product 1c in dichloromethane (20 mL)and the mixture was stirred overnight. Methanol was added to quenchexcess thiophosgene, and the residue after removal of solvent waspurified by silica gel column chromatography eluting with 5%methanol/dichloromethane and further recrystallized fromdichloromethane/hexane to give 130 mg (40%).

¹HNMR (CDCl₃, 300 MHZ) δ 3.79(s, 4H), 3.96(s, 3H), 3.97(s, 3H), 4.07(s,4H), 4.79(s, 2H), 5.92(s, 2H), 6.75(d, J=7.9 Hz, 1H), 6.81(d, J=7.9 Hz,1H), 6.87(s, 1H), 7.04(s, 1H), 7.18(s, 1H), 8.40(s, 1H). FAB HRMS [M+H]⁺calcd for C₂₅H₂₆N₆O₄S: 506.1736; found 507.1820. Combustion Analysis:C₂₅H₂₆N₆O₄S Requires C, 59.27%; H, 5.17%; N, 16.59%; O, 12.63%; S,6.33%. Found C, 59.89%; H, 5.65%; N, 16.99%; O, 12.83%; S, 6.83%.

Example 204-(6,7-Dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioicacid [4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide (2)

To a solution of 6,7-dimethoxy-4-piperazino-9H-pyrimido[4,5-b]indole(200 mg, 0.64 mmol) and pyridine (0.5 mL, 6.4 mmol) in dichloromethane(20 mL) was added a solution of product 1d in dichloromethane (20 mL)and this was stirred overnight. Methanol was added to quench excessthiophosgene, and the residue after removal of solvent was purified bysilica gel column chromatography and eluted with 5%methanol/dichloromethane and further recrystallized fromdichloromethane/hexane to give 50 mg (16%).

¹HNMR (DMSO-d₆, 300 MHZ) δ 3.75(s, 4H), 3.87(s, 3H), 3.88(s, 3H),4.19(s, 4H), 7.04-7.06 (m, 1H), 7.07(s, 1H), 7.24(s, 1H), 7.53(d, J=8.4Hz, 2H), 7.90(d, J=8.4 Hz, 2H), 8.44(s, 1H), 8.51(d, J=4.8HZ, 2H),9.72(s, 1H, —NH), 12.01 (s, 1H, —NH). FAB HRMS [M+H]⁺ calcd forC₂₇H₂₇N₉O₄S₂: 605.1627; found 606.1699. Combustion Analysis: RequiresC₂₇H₂₇N₉ O₄S₂ Requires C, 53.54%; H, 4.49%; N, 20.81%; O, 10.57%; S,10.59%. Found C, 53.84%; H, 4.91%; N, 21.21%; O, 11.87%; S, 8.17%.

Example 214-Benzo[4,5]furo[3,2-d]pyrimidin-4-yl-piperazine-1-carbothioic acid(benzo[1,3]dioxol-5-ylmethyl)-amide (3)

To a solution of 4-piperazinobenzofurano[3,2-d]pyrimidine (200 mg, 0.79mmol) and pyridine (0.5 mL, 7.9 mmol) in dichloromethane (20 mL) wasadded a solution of product 1c in dichloromethane (20 mL) and this wasstirred overnight. Methanol was added to quench excess thiophosgene, andthe residue after removal of solvent was purified by silica gel columnchromatography eluting with 5% methanol/dichloromethane and furtherrecrystallized from dichloromethane/hexane to give 150 mg (37%).

¹HNMR (CDCl₃, 300 MHZ) δ 4.09(s, 4H), 4.27(s, 4H), 4.82(d, J=4.7 Hz,2H), 5.99(s, 2H), 6.77-6.79(m, 1H), 6.80-6.83(m, 1H), 6.89(s, 1H),7.47-7.52(m, 1H), 7.61-7.65(m, 1H), 7.66-7.70(m, 1H), 8.33(d, J=7.0 Hz,1H). FAB HRMS [M+H]⁺ calcd for C₂₃H₂₁,N₅O₃S: 447.1365; found 448.1443.Combustion Analysis: C₂₃H₂₁N₅O₃S Requires C, 61.73%; H, 4.73%; N,15.65%; O, 10.73%; S, 7.17%. Found C, 61.95%; H, 4.99%; N, 15.93%; O,11.13%; S, 7.55%.

Example 224-Benzo[4,5]furo[3,2-d]pyrimidin-4-yl-piperazine-1-carbothioic acid[4-(Pyrimidin-2-ylsulfamoyl)-phenyl]-amide (4)

To a solution of 4-piperazinobenzofurano[3,2-d]pyrimidine (200 mg, 0.79mmol) and pyridine (0.5 mL, 7.9 mmol) in dichloromethane (20 mL) wasadded a solution of product 1d in dichloromethane (20 mL) and this wasstirred overnight. Methanol was added to quench excess thiophosgene; andthe residue after removal of solvent was purified by silica gel columnchromatography eluting with 5% methanol/dichloromethane and furtherrecrystallized from dichloromethane/hexane to give 150 mg (37%).

¹HNMR (DMSO-d₆, 300 MHZ) δ 4.17(s, 8H), 7.04-7.08(m, 1H), 7.49-7.52(m,1H), 7.56-7.59(m, 1H), 7.70-7.75(m, 1H), 7.84(d, J=8.2 Hz, 1H), 7.91(d,J=8.6 Hz, 2H), 8.12 (d, J=7.6 Hz, 2H), 8.52(d, J=4.8 Hz, 2H), 8.58(s,1H), 9.82(s, 1H, NH). FAB HRMS [M+H]⁺ calcd for C₂₅H₂₂N₈O₃S₂: 546.1256;found 547.1325. Combustion Analysis: C₂₅H₂₂N₈O₃S₂ Requires C, 54.93%; H,4.06%; N, 20.50%; O, 8.78%; Si 1.73%. Found 55.35%; H, 4.44%; N, 20.83%;O, 8.96%; S, 11.89%.

Example 23 4-(9-Thia-1,5,7-triaza-fluoren-8-yl)-piperazine-1-carbothioicacid (benzo[1,3]dioxol-5-ylmethyl)-amide (5)

To a solution of 4-piperazinopyrido[3′,2′;4,5]thieno[3,2-d]pyrimidine(200 mg, 0.74 mmol) and pyridine (0.5 mL, 7.9 mmol) in dichloromethane(20 mL) was added a solution of product 1c in dichloromethane (20 mL)and this was stirred overnight. Methanol was added to quench excessthiophosgene, and the residue after removal of solvent was purified bysilica gel column chromatography eluting with 5%methanol/dichloromethane and further recrystallized fromdichloromethane/hexane to give 110 mg (32%).

¹HNMR (CDCl₃, 300 MHZ) δ 4.07(s, 4H), 4.17(s, 4H), 4.72(d, J=4.5 Hz,2H), 5.88(s, 2H), 6.69(d, 1H), 6.75(d, 1H), 6.80(s, 1H), 7.43-7.47(m,1H), 8.65(s, 1H), 8.75(d, J=3.8 Hz, 2H). FAB HRMS [M+H]⁺ calcd forC₂₂H₂₀N₆O₂S₂: 464.1089; found 465.1167. Combustion Analysis:C₂₂H₂₀N₆O₂S₂ Requires C, 56.88%; H, 4.34%; N, 18.09%; O, 6.80%; S,13.80%. Found C, 57.16%; H, 4.94%; N, 18.53%; O, 6.97%; S, 14.30%.

Example 24 4-(9-Thia-1,5,7-triaza-fluoren-8-yl)-piperazine-1-carbothioicacid [4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide (6)

To a solution of 4-piperazinopyrido[3′,2′;4,5]thieno[3,2-d]pyrimidine(200 mg, 0.74 mmol) and pyridine (0.5 mL, 7.9 mmol) in dichloromethane(20 mL) was added a solution of product 1d in dichloromethane (20 mL)and this was stirred overnight. Methanol was added to quench excessthiophosgene, and the residue after removal of solvent was purified bysilica gel column chromatography eluting with 5%methanol/dichloromethane and further recrystallized fromdichloromethane/hexane to give 60 mg (15%).

¹HNMR (DMSO-d₆, 300 MHZ) δ 4.07(s, 8H), 6.96-6.99(m, 1H), 7.47-7.50(m,1H), 7.58-7.62(m, 1H), 7.82(d, J=8.6 Hz, 2H), 8.43(d, J=4.9 Hz, 2H),8.63 (d, J=8.02 Hz, 2H), 8.70(s, 1H), 8.80(d, J=4.0 Hz, 1H). FAB HRMS[M+H]⁺ calcd for C₂₄H₂₁N₉O₂S₃: 563.0980; found 564.1059. CombustionAnalysis: C₂₄H₂₁N₉O₂S₃ Requires C, 51.14%; H, 3.76%; N, 22.36%; O,5.68%; S, 17.07%. Found 51.44%; H, 3.98%; N, 22.84%; O, 5.96; S, 17.45.

Example. 25 4-(6,7-Dimethoxy-quinazolin-4-yl)-piperazine-1-carbothioicacid [4-(Pyrimidin-2-ylsulfamoyl)-phenyl]-amide (7)

To a solution of 4-(1-piperazinyl)-6,7-dimethoxy quinazoline (200 mg,0.73 mmol) and pyridine (0.5 mL, 6.4 mmol) in dichloromethane (20 mL)was added a solution of product 1d in dichloromethane (20 mL) and thiswas stirred overnight. Methanol was added to quench excess thiophosgene,and the residue after removal of solvent was purified by silica gelcolumn chromatography eluting with 5% methanol/dichloromethane andfurther recrystallized from dichloromethane/hexane to give 80 mg (20%).

¹HNMR (CDCl₃, 300 MHZ) δ 3.85(s, 4H), 3.98(s, 3H), 4.02(s, 3H), 4.11(s,4H), 6.98(m, 1H), 7.08(s, 1H), 7.32(d, 2H), 7.88(s, 1H), 8.00(d, J=6.7Hz, 2H), 8.62(d, 2H), 8.66(s, 1H). FAB HRMS [M+H]⁺ calcd forC₂₅H₂₆N₈O₄S₂: 566.1518; found 567.1597. Combustion Analysis:C₂₅H₂₆N₈O₄S₂ Requires C, 52.99%; H, 4.62%; N, 19.77%; O, 11.29%; S,11.32%. Found C, 53.27%; H, 4.94%; N, 19.99%; O, 11.57%, S, 11.64%.

Example 26 Cancer Cell Cytotoxicity Assay

To validate the hypothesis that the designed c-kit/PDGFR tyrosine kinaseinhibitors mediate GIST882 cell killing and PDGFR-mediated cell killingof pancreatic cancer cell lines (CFPAC-1, PANC-1 and MIA PaCa-2), an invitro cytotoxicity assay was performed. The GIST882 cell line used inthis study has a c-kit gain-of-function mutation (K642E). The assayutilized the Cell Titer 965 Non-Radioactive Cell Proliferation Assay(Promega Corp., Madison, Wis.). First the cells were cultured. GIST882cells were provided by Dr. Jonathan A. Fletcher (Dana-Farber CancerInstitute, Boston, Mass.). PANC-1 and MIAPaCa-2 cells were provided byDr. Daniel Von Hoff (Arizona Cancer Center, Tucson, Ariz.). GIST882cells were cultured in RPMI 1640 medium (Cat# 21870-076, InvitrogenCorporation) supplemented with 300 mg/L L-glutamine, 100 unit/mlpenicillin, 100 μg/ml streptomycin and 15% fetal bovine serum. PANC-1and MIAPaCa-2 cells were maintained in RPMI 1640 medium (cat# 10-040,Mediatech, Inc.) supplemented with 100 unit/ml penicillin, 100 μg/mlstreptomycin and 10% fetal bovine serum. All the cell lines wereincubated in a humidified incubator at 37° C. with 5% CO₂ atmosphere.

Cells were plated at a density of 2000 to 10000 cells per well,depending on their growth rate, in 0.1 mL medium on day 0 in 96-wellFalcon microtiter plates (#3072, Becton-Dickinson Labware, Lincoln Park,N.J.). On day 1, 10 μL of serial dilutions of the individual compoundswere added to the plates in replicates of 4. After incubation for 4 daysat 37° C. in a humidified incubator, the cells were fixed with 10%Trichloroacetic acid solution (Catalog No. 490-10, Sigma). Subsequently,they were labeled with 0.04% Sulforhodamine B (S9012, Sigma) in 1%acetic acid. After multiple washes to remove excess dye, 100 μl of 50 mMTris solution was added to each well in order to dissolve the dye. Theabsorbance of each well was read on a plate reader (Wallac Vector²,PerkinElmer) at the wavelength of 570 nm. Data were expressed as thepercentage of survival of control calculated from the absorbancecorrected for background absorbance. The surviving percent of cells wasdetermined by dividing the mean absorbance values of the monoclonalantibody by the mean absorbance values of the control and multiplying by100.

The calculated FlexX scoring and IC₅₀ values for these novel and priorart c-kit inhibitors are shown in Table 2 below. Not all of the novelcompounds evaluated exhibited cytotoxicity against GIST882 cells.Moreover, in an in vitro assay of aurora 2 kinase, a serine/threoninekinase, these compounds showed no activity (data not shown). Takentogether, these results validate compounds of the invention, such asHPK61 (II-2-7) and HPK56 (III-1-3), as potent, specific c-kit and PDGFRtyrosine kinase inhibitors.

A comparison of the cytotoxicity profiles of the designed andsynthesized compounds 1-7 (FIG. 17), as well as known kinase inhibitorsSTI571 and CT52923, is shown in FIGS. 22A, 22B and 22C, and thecalculated IC₅₀ values are shown below in Table 2. For the GIST882 cellline, HPK61 (II-2-7), HPK56 (III-1-3), STI571, and CT52923 weresimilarly potent, with IC₅₀ values ranging from 0.1 to 1.8 μM and with apotency order of STI571 (0.1 μM)>HPK61 (II-2-7)(0.45 μM)>HPK56(III-1-3)(1.60 μM)>CT52923 (1.80 μM). Although STI571 killed cellsearly, 25% of cells exposed to STI571 were alive at day 4. In contrast,HPK61 (II-2-7) and HPK56 (III-1-3) had a more prolonged effect, with 5%of cells alive at day 4. For the pancreatic cancer cell lines MIAPaCa-2and PANC-1, HPK56 (III-1-3) was the most potent, with IC₅₀ values of2.10 and 3.00 μM, respectively, and a potency order of HPK56(III-1-3)(2.1-3.0 μM)>HPK61 (II-2-7)(15.5-16.0 μM)>STI571 (20.0-24.0μM)>CT52923 (25.0-26.6 μM).

TABLE 2 Activity (IC₅₀ μM) and FlexX (kJ/mol) results of lead compoundsand tricyclic and quinazoline inhibitors against c-kit and PDGFRtyrosine kinases. PDGFR FlexX^(a) Struc- c-kit PANC- FlexX Drug Compoundture GIST882 MIAPaCa 1 score score 1 (HPK61) II-2-7 0.45 15.5 16.0 −34.8−66.9 2 (HPK62) II-2-6 28.0 >50 >50 −19.3 −44.5 3 (HPK56) III-1-3 1.602.10 3.00 −28.4 −62.4 4 (HPK59) III-1-5 27.5 ND^(b) ND −27.9 −59.3 5(HPK57) III-1-4 28.0 >50 >50 −22.2 −54.3 6 (HPK60) 34-4 50.0 >50 >50−21.1 −57.2 (Table 4) 7 (HPK16) IV-1-3 50.0 >50 >50 −21.2 −50.6^(a)FlexX score for c-kit tyrosine kinase. FlexX belongs to the categoryof empirical free energy scoring function (energy decomposition intovarious scores to which a coefficient has been assigned). The drug scorecombines drug likeness, cLogP, molecular weight, and toxicity risks inone handy value than may be used to judge the compound's overallpotential to qualify for a drug. ^(b)ND: not determined. ^(c) NA: notavailable.

Furthermore, a recent study reported that approximately 35% of GISTsamples lacked c-kit mutations and had activation mutations in PDGFR-A(Heinrich, M., et al., Science 299(5607):708-10, 2003). Docking studiesdemonstrated that HPK61 (II-2-7) and HPK56 (III-1-3) interact equallywith the tyrosine kinase domains of c-kit and PDGFR. Cellularcytotoxicity assays demonstrated that HPK61 (II-2-7) and HPK56 (III-1-3)are highly selective for c-kit and PDGFR tyrosine kinases and aresuperior to STI571 and CT52923 in pancreatic cancer cell lines.Therefore, it is expected that HPK61 (II-2-7) and HPK56 (III-1-3), aswell as other related compounds of the invention, will be effective intreating both c-kit- and PDGFR-mediated GIST.

Example 27 Kinase Inhibition Assay

This example describes the inhibitory activity of compound (II-2-6),also referred to herein as HPK62), against various kinase proteins,including Aurora-A, cAMP-PK, MKK6 and CDK1.

In vitro enzyme assays were performed using the Kinase-Glo™ LuminescentKinase Assay from Promega Corporation (Madison, Wis.). The followingconditions were used:

Kinase Enzyme [ATP] (μM) Substrate [Substrate] (μM) Aurora-A  20 ng 0.1Kemptide 30 cAMP-PK 0.5 units 0.1 Kemptide 30 MKK6 1.0 μg 0.1 Kemptide30 CDK1  10 units 0.1 Kemptide 30

Enzymatic reactions were allowed to progress for 2 hours at 30° C., thenassayed for kinase activity according to manufacturer protocol. Thefollowing IC₅₀ values were determined for the compound, using the abovekinases:

Kinase IC₅₀ (μM) Aurora-A 0.9 cAMP-PK >100 MKK6 6.2 CDK1 22.3

Example 28 Effects of Compound (II-2-6) on Cell Cycle Distribution

The effects of Structure (II-2-6) on cell cycle distribution wereassayed using flow cytometry, using the following procedure: MIA PaCa-2cells (American Type Culture Collection, Manassas, Va.) were grown to˜40% confluency. At this point, MP-235 at various concentrations, or anequal volume of DMSO (drug diluent) was added. Cells were grown in thepresence of drug for 48 hours, and harvested using trypsin. 1 millioncells were washed in 1 mL of Modified Krishan's Buffer (0.1% sodiumcitrate, 0.3% NP-40, 0.05 mg/ml propidium iodide, 0.02 mg/ml RNase A),and resuspended in 1 mL of fresh Modified Krishan's Buffer. Cell pelletswere kept at 4° C. for no more than 24 hours before flow cytometricanalysis was performed by the University of Arizona Flow Cytometry CoreFacility. The cell cycle profile obtained from this analysis isillustrated in FIG. 23.

Example 29 Effects of Compound (II-2-6) on Cell Proliferation

The ability of compound (II-2-6) at various concentrations to inhibitcell proliferation was also tested, using the MIA PaCa-2 cell line.200,000 MIA PaCa-2 cells were plated into each well of a six-well plateand incubated overnight. At this point, MP-235 at variousconcentrations, or an equal volume of DMSO (drug diluent) was added.Cells were grown in the presence of drug for 48 hours, and harvestedusing trypsin. The number of cells in each well was determined by a cellcounting assay using a hematocytometer. Each drug concentration wastested in triplicate and each well was counted in triplicate. Reductionin cell proliferation was determined by dividing the number of cells indrug-treated wells by the number in equivalent DMSO-treated wells.Results from this analysis are illustrated in FIG. 24.

Example 30 Effects of Structure (II-2-6) on Cytotoxicity of PancreaticCancer Cell Lines

To determine if the reduction in cell number was due to slowing of cellgrowth or outright cell killing, the cytotoxicity of Structure (II-2-6)was determined, using an MTS-based assay in cultured MIA PaCa-2 andPanc-1 pancreatic cancer cells. In vitro cytotoxicity assays wereperformed using the CellTiter 96 Non-Radioactive Cell ProliferationAssay (Promega Corp., Madison, Wis.). Cells were plated in 0.1 ml mediumon day 0 in 96-well microtiter plates (Falcon, #3072). On day 1, 10 μLof serial dilutions of the test agent were added in replicates of 4 tothe plates. After incubation for 4 days at 37° C. in a humidifiedincubator, 20 μl of a 20:1 mixture of[3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS], 2 mg/ml, and an electron coupling reagent, phenazinemethosulfate (PMS, 0.92 mg/ml in DPBS), was added to each well andincubated for 1 or 2 hours at 37° C. Absorbance was measured using Model7520 microplate reader (Cambridge Technology, Inc.) at 490 nm. Data wereexpressed as the percentage of survival of control calculated from theabsorbance corrected for background absorbance. The surviving fractionof cells was determined by dividing the mean absorbance values of thetest agents by the mean absorbance values of untreated control. Platereadings at 490 nm were taken after 60 and 120 minutes of incubationwith the MTS substrate, and the results are illustrated in FIGS. 25A-B,respectively.

Example 31 Effects of Compound (II-2-6) on Cytotoxicity of Colon,Breast, Ovarian and Pancreatic Cancer Cell Lines

These cytotoxicity data were further complemented by performing the sameMTS assay described above in a number of different cell lines fromvarious sources. The results obtained from these experiments areillustrated in FIGS. 26A-C.

Example 32 Further Illustrative Inhibitory Compounds

Compound (II-2-6) is an illustrative kinase inhibitory compound of theinvention belonging to a class of 4-Piprazinylpyrimido[4,5-b]indoles.This series of compounds was designed as inhibitors of both aurora-2 andc-kit kinases and Structure (II-2-6) was confirmed to have low nanomolarinhibitory activity against Aurora-2 kinase and to have low AMinhibitory activity against c-kit kinase.

Compound (II-2-6) analogues were designed and synthesized according toSchemes 3-5 below in order to evaluate and optimize aurora-2 kinaseactivity, aqueous solubility and pharmacokinetic/pharmacodynamicprofiles. The compounds belong to the class of pyrimido[4,5-b]indoles(Ia to Id) and quinazolines (IIa to IId below). Detailed structuralinformation of illustrative compounds is provided in Table 3 below.Analogues were made in which R₁, R₂, R₃ and R₄(1)-4-Piprazinylpyrimido[4,5-b]indoles, pyrimido[4,5-b]indoles offormula Ia-Id and R₁, R₂, R₃ and R₄ (1)-4-piperazin-1-yl-quinazolinesand substituted quinazoline compounds of IIa-IId were synthesized.

Based on the docking results, (II-2-6) binds to the ATP-binding pocketand is involved in several Van der Waals contacts and hydrogen bondinginteractions with the active site pocket. The 6,7-dimethoxypyrimido[4,5-b]indole moiety positioned into the adenine binding pocket,the 6,7-substituents of the pyrimido[4,5-b]indole orients from the hingeregion into the solvent pocket and the benzenesulfonamide group isinvolved in interactions with the β and γ phosphate regions, whereas thepiprazine group occupies the sugar binding pocket. Structure (II-2-6)had strong hydrogen bonding interactions with Pro214, Arg220 and is inclose contact with Glu211 and Ala213 residues. The sulfonamide —S═Ogroup forms hydrogen bonds with Lys258. In terms of hydrophobicity,areas deep in the ATP pocket around Phe144 are occupied by the flataromatic ring and pyrimidine ring of (II-2-6).

Several analogues of (II-2-6) were studied using virtual docking topredict their binding mode. The compounds developed based on the mode ofbinding of (II-2-6) were undertaken for synthesis. Synthetic approachesfor generating substitutions at R₁, R₂, R₃, R4, R₅, R₆ and X are setforth in the following Schemes 3 to 7, and illustrative compounds aredepicted in Table 3.

TABLE 3 No Structure* 32-1

32-2

32-3

32-4

32-5

32-6

32-7

32-8

32-9

32-10

32-11

32-12

32-13

32-14

32-15

32-16

32-17

32-18

32-19

32-20

32-21

32-22

32-23

32-24

32-25

32-26

32-27

32-28

32-29

32-30

32-31

32-32

32-33

32-34

32-35

32-36

32-37

32-38

32-39

32-40

32-41

32-42

32-43

32-44

32-45

32-46

32-47

32-48

32-49

32-50

32-51

32-52

32-53

32-54

32-55

32-56

32-57

32-58

32-59

32-60

32-61

32-62

32-63

32-64

32-65

32-66

32-67

32-68

32-69

32-70

32-71

32-72

32-73

32-74

32-75

32-76

32-77

32-78

32-79

32-80

32-81

32-82

32-83

32-84

32-85

32-86

32-87

32-88

32-89

32-90

32-91

32-92

32-93

32-94

32-95

32-96

32-97

32-98

32-99

32-100

32-101

32-102

32-103

32-104

32-105

32-106

32-107

32-108

32-109

32-110

32-111

32-112

32-113

32-114

32-115

32-116

32-117

Example 33 Compound (II-2-6) Protein Kinase Inhibitory Activity

The protein serine-threonine kinases cAMP PK, MKK6 and Cdk1 were testedalongside Aurora-2 kinase to evaluate the activity of compound (II-2-6)against these protein kinases. Briefly, in this assay kinase activity isdetermined by quantifying the amount of ATP remaining in solutionfollowing the kinase reaction by measuring the relative light units(RLU) produced by luciferase using a luminometer. Percent activity wasdetermined for individual compounds by comparing luminometer readings ofdrug-treated reactions to controls containing no drug (RLU_(NO Inhib))and no Aurora-2 enzyme (RLU_(NO Kinase)) in the following equation:

${{Percent}\mspace{14mu}{I{nhibition}}} = {\frac{{R{LU}}_{{No}\mspace{14mu}{Kinase}} - {RLU}_{drug}}{{RLU}_{{No}\mspace{14mu}{Kinase}} - {RLU}_{{No}\mspace{14mu}{Inhib}}} \times 100}$

In a 50 μl reaction, 20 ng of recombinant aurora-2 kinase (Upstate, LakePlacid, N.Y.) was incubated at 30° C. for two hours with shaking (360rpm) with 62.5 μM Kemptide (Calbiochem, San Diego, Calif.), 3 μM ATP(Invitrogen, Carlsbad, Calif.) and kinase reaction buffer (40 mMTris-HCl, 20 mM MgCl₂ and 0.1 μg/μl bovine serum albumin). The value of3 μM ATP was determined to be the Km (concentration at which the enzymeis working at 50% maximum velocity) for the amount of enzyme used inthis assay. This reaction was carried out in the presence of drugsubstances, which had been previously diluted to desired concentrationsin DMSO. After incubation, 50 μl of Kinase-Glo® (Promega, Inc., Madison,Wis.) solution was added to each reaction mixture and allowed toequilibrate for 10 minutes at room temperature. Kinase-Glo solutioncontains luciferase enzyme and luciferin, which react with ATP toproduce light. Kinase activity is determined by quantifying the amountof ATP remaining in solution following the kinase reaction by measuringthe relative light units (RLU) produced by luciferase using aluminometer (Thermo Electron Corporation, Vantaa, Finland).

The results of these experiments are shown in FIG. 27. Compound (II-2-6)had inhibitory activity against each of the kinases tested, with highestactivity against Aurora-2 kinase.

Example 34 Synthesis and Analysis of Further Illustrative Compounds

Compound (III-1-3), also referred to herein as HPK56/MP-470, is anillustrative compound of the present invention having the followingstructure:

Analogues of (III-1-3) were designed and synthesized in order toevaluate and optimize kinase selectivity, aqueous solubility, and toimprove pharmacokinetic and pharmacodynamic profiles. Illustrativesynthesis approaches for generating (III-1-3) analogues are depicted inthe synthesis schemes below. Synthesis of R₁ substitutedbenzofuranopyrimidines was undertaken. The methyl3-guanidinobenzofuran-2-carboxylate is prepared from methyl3-aminobenzofuran-2-carboxylate by reacting with cyanoacetamide inpresence of dioxane and dry HCl gas. The obtained guanidine is cyclizedin the presence of aqueous NaOH. Similar procedures were utilized forpreparing 2-substituted (III-1-3) and its analogues as depicted in theSchemes 8-10 set forth below. Introduction of —NH₂ at the 2 position wasutilized for various sulfonic, inorganic and hydroxyacid salts.Illustrative compounds are shown in Table 4 below.

TABLE 4 No Structure 34-1

34-2

34-3

34-4

34-5

34-6

34-7

34-8

34-9

34-10

34-11

34-12

34-13

34-14

34-15

Example 35 Analysis of Compound Binding and Inhibitory Activity againstc-kit Mutants

The published crystal structure of c-kit kinase (pdb code:1PKG) and itsmutated structure were used to study the mode of binding of compound(III-1-3) (HPK56/MP-470), a benzofuranopyrimidine compound, its2-substituted analogs, and quinazoline derivatives.

All molecular modeling studies including docking were carried out usingSCHRÖDINGER software (SCHRÖDINGER L.L.C, New York) running on RedHatLinux. The published crystal structure of c-kit kinase (1) was used forprotein preparation, generation of grids and docking using a program,Glide, which is implemented in the SCHRÖDINGER software.

The c-kit mutations in GIST tumors and their interactions with (III-1-3)and its analogues were studied on wild type c-kit, K642E (an exon 13mutant) and D816V (an exon 17 mutant). Glide scores were generated foreach compound for both wild-type and c-kit mutants. A more negativeglide score is predictive of stronger binding. The determined Glidescores are shown below in Table 5. The mode of binding of (III-1-3) withthese mutated c-Kit proteins predicts that (III-1-3) is more effectivein binding both K642E and D816V mutations relative to wild-type c-kit.

Table 5 also shows IC₅₀ values in the GIST882 cell line determined forthe same compounds. Briefly, cells are seeded into 96-well,tissue-culture treated, opaque white plates (Thermo Electron, Vantaa,Finland), at between 5000 and 7500 cells per well, depending on thespeed of cell proliferation, in 100 μl of appropriate growth medium(determined by the ATCC). Cells are then exposed to the appropriateconcentration of drug or an equal amount of DMSO (drug diluent) andallowed to grow in its presence for 96 hours. Following this, 100 μofCell-Titer-Glo reagent (Promega, Inc., Madison, Wis.) is added to eachwell. Plates are then shaken for 2 minutes at room temperature to allowfor cell lysis and incubated for 10 minutes to stabilize the luminescentsignal. Similar to the Kinase-Glo assay reagent, this reagent containsboth luciferase enzyme and its substrate luciferin. Luciferase,activated by ATP in the cell lysate, catalyzes the conversion ofluciferin to oxyluciferin, a reaction which produces light. The amountof light produced is proportionate to the amount of ATP in the celllysate, which is itself proportional to cell number and gives an indexof cellular proliferation. The IC₅₀ is defined as the concentration ofdrug that yields a 50% inhibition of cell growth, as compared to wellscontaining untreated cells.

TABLE 5 Activity (IC₅₀ μM) and Glide score results of inhibitors againstWT and mutated c-kit tyrosine kinases. Glide score GIST882 K642E/Compound Structure IC₅₀ (μM) WT K642E D816V D816V HPK61 II-2-7 0.45−9.20 −8.79 −8.93 −9.10 HPK62 II-2-6 28.0 −7.13 −6.39 −6.42 −6.22 HPK56III-1-3 1.60 −8.83 −9.96 −10.43 −10.19 (MP470) HPK59 III-1-5 27.5 −7.24−7.01 −6.89 −6.37 HPK57 III-1-4 28.0 −6.53 −6.21 −6.49 −6.89 HPK60 34-450.0 −6.65 −6.60 −6.53 −6.52 (Table 4) HPK16 IV-1-3 50.0 −6.98 −7.21−7.43 −7.89

Example 36 Kinase Inhibitory Activity of Compounds (III-1-3) and(II-2-7)

Compounds (III-1-3) and (II-2-7) are illustrative compounds of thepresent invention having the structures shown below:

These compounds were tested for their inhibitory activity against c-Kitand the related receptor tyrosine kinase, PDGFRa. Enzymes were incubatedwith the appropriate concentration of inhibitor and radiolabeledγ-³²P-ATP. After 30 minutes, the reaction mixtures were electrophoresedon an acrylamide gel and autophosphorylation, quantitated by the amountof radioactivity incorporated into the enzyme, was assayed. Results fromthese experiments are shown in FIGS. 28A and 28B Both (III-1-3) and(II-2-7) demonstrated dose-dependent c-kit inhibitory activity againstc-Kit and PDGRFa.

Example 37 Inhibitory Activity of Additional Illustrative Compounds

Various compounds of the invention, including (IV-1-3) (also referred toas HPK16), (III-1-3) (also referred to as HPK56), (III-1-4) (alsoreferred to as HPK57), (III-1-5) (also referred to as HPK59), and(II-2-7) (also referred to as HPK61) were tested for activity againstGIST tumor cells using the GIST882 cell line. Briefly, cells are seededinto 96-well, tissue-culture treated, opaque white plates (ThermoElectron, Vantaa, Finland), at between 5000 and 7500 cells per well,depending on the speed of cell proliferation, in 100 μl of appropriategrowth medium (determined by the ATCC). Cells are then exposed to theappropriate concentration of drug or an equal amount of DMSO (drugdiluent) and allowed to grow in its presence for 96 hours. Followingthis, 100 μl of Cell-Titer-Glo reagent (Promega, Inc., Madison, Wis.) isadded to each well. Plates are then shaken for 2 minutes at roomtemperature to allow for cell lysis and incubated for 10 minutes tostabilize the luminescent signal. Similar to the Kinase-Glo assayreagent, this reagent contains both luciferase enzyme and its substrateluciferin. Luciferase, activated by ATP in the cell lysate, catalyzesthe conversion of luciferin to oxyluciferin, a reaction which produceslight. The amount of light produced is proportionate to the amount ofATP in the cell lysate, which is itself proportional to cell number andgives an index of cellular proliferation. The IC₅₀ is defined as theconcentration of drug that yields a 50% inhibition of cell growth, ascompared to wells containing untreated cells. The results of theseexperiments are shown in FIG. 29, demonstrating that all of thecompounds tested had dose-dependent inhibitory activity, while HPK56(III-1-3) and HPK61 (II-2-7) had the highest inhibitory activity of theinventive compounds tested.

Example 38 Synthesis of Additional Illustrative Protein KinaseInhibitors

The following example describes the synthesis of the illustrativecompounds of the present invention set forth below in Table 6, using thegeneral synthesis Schemes 11-15 also shown below. The synthesis methodsbelow are illustrative in nature and can be readily modified usingroutine and established principles of synthetic organic chemistry toproduce the inventive compounds described herein.

All experiments were carried out under an inert atmosphere and at refluxand or room temperature unless otherwise stated. The purities ofcompounds were assessed by routine analytical HPLC. TLCs were performedon precoated silica gel plates (Merck), and the resulting chromatogramswere visualized under UV light at 254 nm. Melting points were determinedon a Kofler Block or with a Büchi melting point apparatus on compoundsisolated as described in the experimental procedures and areuncorrected. The NMR spectra were determined in DMSO-d₆ solution (unlessotherwise stated) on a Bruker AM 300 (300 MHz) spectrometer or on aVarian 400 (400 MHz). Chemical shifts are expressed in unit of 67 (ppm),and peak multiplicities are expressed as follows: s, singlet; d,doublet; dd, doublet of doublet; t, triplet; br s, broad singlet; m,multiplet. FAB measurements have been carried out on a mass spectrometerHX-110 instrument (JEOL, Akishima, Japan) equipped with a conventionalXe gun. A mixture matrix of glycerol:thioglycerol:mNBA (meta-nitrobenzylalcohol) 50:25:25 containing 0.1% of trifluoroacetic acid (TFA) wasused. For accurate mass measurements, polyethylene glycol (PEG) was usedas the internal standard. Combustion analysis (CHNS) was performed byDesert Analytics Laboratory, Tucson, Ariz.

TABLE 6 No Structure 38-1

38-2

38-3

38-4

38-5

38-6

38-7

38-8

38-9

38-10

38-11

38-12

38-13

38-14

38-15

38-16

38-17

A.4-(6,7-dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioicacid [4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide (1). (see Scheme 11)

7-dimethoxy-4-(piperazin-1-yl)-9H-pyrimido[4,5-b]indole 8 in DCM wasadded dropwise to compound 10 in DCM over a period of 15 minutesfollowed by the addition of excess pyridine. The resulting reactionmixture was stirred at RT for 24 hours. After the completion of thereaction, MeOH was added to quench the excess of compound 10 and thesolvents were evaporated. The crude product was purified by columnchromatography using a DCM/5% MeOH solvent system. The obtained product1 (Table 6) (compound 9 in Scheme 11) is a half white solid with a yieldof 37.6%.

¹HNMR (DMSO-d₆, 300 MHz): δ 3.75(s, 4H), 3.87(s, 3H), 3.88(s, 3H),4.19(s, 4H), 7.04-7.06 (m, 1H), 7.07(s, 1H), 7.24(s, 1H), 7.53(d, J=8.4Hz, 2H), 7.90(d, J=8.4 Hz, 2H), 8.44(s, 1H), 8.51(d, J=4.8 HZ, 2H),9.72(s, 1H, —NH), 12.01(s, 1H, —NH). FAB HRMS [M+H]⁺ calcd forC₂₇H₂₇N₉O₄S₂: 605.1627; found 606.1699.

B. 7-dimethoxy-4-(piperazin-1-yl)-9H-pyrimido[4,5-b]indole:

4-Chloro-6,7-dimethoxy-9,9a-dihydro-4aH-pyrimido[4,5-b]indole 7 wasdissolved in p-dioxane (50 mL), and piprazine (3.9 g) was addedfollowing the addition of pyridine (5 mL) under argon at RT. Thereaction mixture was heated to reflux for 16 hours and it was cooled.The solvents were removed under vacuum and the obtained crude productwas purified by flash coloumn chromatograph using a DCM/10% MeOH solventsystem. The compound 8 obtained after purification yielded 66% (3.9 g)as half white solid.

C. 4-Chloro-6,7-dimethoxy-9,9a-dihydro-4aH-pyrimido[4,5-b]indole:

A suspension of 6,7-dimethoxy-3H-pyrimido[4,5-b]indol-4(9H)-one 6 (2.8g), POCl₃ (20 mL) and p-dioxane 65 mL was heated at reflux for 6 hrs,then stirred at 250° C. for 36 hrs. The obtained mixtrure was filteredand concentrated. The crude product was purified by columnchromatography using 1% MeOH/DCM to give title compound 7 73.3% (2.2 g)as pale yellow solid.

D. [4-(Pyrimidin-2-ylsulfamoyl)-phenyl]-thiophosgene chloride:

Thiophosgene (0.78 mL) was slowly added to the stirred solution ofsulfadiazine (1.71 g) in DCM (50 mL) following the addition oftriethylamine (0.47 mL) at 0° C. After the additions, the reactionmixture was stirred at RT for 5 hrs. The reaction mixture is dilutedwith more DCM and is washed with water and brine and the obtainedsolvent was dried over Na₂SO₄. Solvent is evaporated and dried undervacuum to give compound 15 (Scheme 12) as yellowish orange solid in64.5% yield and it was used directly in the next step.

E.N-(4-{[4-(6,7-Dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbo-thioyl]-amino]-phenyl)-benzamide(2)

¹HNMR (DMSO d6, 300 MHZ) 3.73(s, 4H), 3.87(d, 6H, J=5.6 Hz), 4.17 (s,4H), 7.06(s, 1H), 7.25(d, 2H, J=6.4 Hz), 7.29(s, 1H), 7.55(m, 3H),7.70(d, 2H, J=8.8 Hz), 7.94(d, 2H, J=8.0 Hz), 8.42(s, 1H), 9.44 (s, 1H,br), 10.24 (s, 1H, br), 11.98 (s, 1H, br). FAB HRMS [M+H]⁺ calcd forC₃₀H₃₀N₇O₃S: 568.6793; found 568.2131.

F.N-(5-{[4-(6,7-Dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbo-thioyl]-amino]-pyridin-2yl)-benzamide(3). ¹HNMR (DMSO d6, 300 MHZ) 3.72(s, 4H), 3.84(d, 6H, J=7.0 Hz), 4.04(s, 4H), 7.05(s, 1H), 7.16(s, 1H), 7.54(m, 3H), 8.03(d, 2H, J=7.4 Hz),8.15(s, 1H), 8.19(d, 2H, J=8.0 Hz), 8.41 (s, 1H), 10.94 (s, 1H, br),11.99 (s, 1H, br). FAB HRMS [M+H]⁺ calcd for C₂₉H₂₉N₈O₃S: 569.2135;found 569.0235. G.N-(5-{[4-(6,7-Dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbo-thioyl]-amino]-pyrimidin-2yl)-benzamide(4).

¹HNMR (DMSO d6, 300 MHZ) 3.80(s, 4H), 3.86(d, 6H, J=7.0 Hz), 4.25 (s,4H), 7.08(s, 1H), 7.27(s, 1H), 7.59(m, 3H), 7.97(d, 2H, J=7.4 Hz),8.46(s, 1H), 8.67(s, 2H), 9.67 (s, 1H, br), 11.01 (s, 1H, br), 12.01 (s,1H, br). FAB HRMS [M+H]⁺ calcd for C₂₈H₂₈N₉O₃S: 570.6548; found570.2027.

H. Acetic acid7-methoxy-4-{4-[4-(pyrimidin-2-ylsulfamoyl)-phenylthio-carbamoyl]-piperazin-1-yl}-9H-pyrimido[4,5-b]indol-6-ylester (5) ¹HNMR (DMSO-d6, 400 MHz) MS [+ve ESI] for C₂₈H₂₇N₉O₅S₂: found634.7012 (M+H)⁺ I.4-Benzo[4,5]furo[3,2-d]pyrimidin-4-yl-piperazine-1-carbothioic acid[4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide (6).

¹HNMR (DMSO-d₆, 300 MHZ) δ 4.17(s, 8H), 7.04-7.08(m, 1H), 7.49-7.52(m,1H), 7.56-7.59(m, 1H), 7.70-7.75(m, 1H), 7.84(d, J=8.2 Hz, 1H), 7.91(d,J=8.6 Hz, 2H), 8.12 (d, J=7.6 Hz, 2H), 8.52(d, J=4.8 Hz, 2H), 8.58(s,1H), 9.82(s, 1H, NH). FAB HRMS [M+H]⁺ calcd for C₂₅H₂₂N₈O₃S₂: 546.1256;found 547.1325.

J. 4-(9-Thia-1,5,7-triaza-fluoren-8-yl)-piperazine-1-carbothioic acid[4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide (7).

¹HNMR (DMSO-d₆, 300 MHZ) δ 4.07(s, 8H), 6.96-6.99(m, 1H), 7.47-7.50(m,1H), 7.58-7.62(m, 1H), 7.82(d, J=8.6 Hz, 2H), 8.43(d, J=4.9 Hz, 2H),8.63 (d, J=8.02 Hz, 2H), 8.70(s, 1H), 8.80(d, J=4.0 Hz, 1H). FAB HRMS[M+H]⁺ calcd for C₂₄H₂₁N₉O₂S₃: 563.0980; found 564.1059.

K. 4-Benzo[4,5]furo[3,2-d]pyrimidin-4-yl-piperazine-1-carbothioic acid(benzo[1,3]dioxol-5-ylmethyl)-amide (8).

¹HNMR (CDCl₃, 300 MHZ) δ 4.09(s, 4H), 4.27(s, 4H), 4.82(d, J=4.7 Hz,2H), 5.99(s, 2H), 6.77-6.79(m, 1H), 6.80-6.83(m, 1H), 6.89(s, 1H),7.47-7.52(m, 1H), 7.61-7.65(m, 1H), 7.66-7.70(m, 1H), 8.33(d, J=7.0 Hz,1H). FAB HRMS [M+H]⁺ calcd for C₂₃H₂₁N₅O₃S: 447.1365; found 448.1443.

L.4-(6,7-Dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioicacid (benzo[1,3]dioxol-5-ylmethyl)-amide (9).

¹HNMR (CDCl₃, 300 MHZ) δ 3.79(s, 4H), 3.96(s, 3H), 3.97(s, 3H), 4.07(s,4H), 4.79(s, 2H), 5.92(s, 2H), 6.75(d, J=7.9 Hz, 1H), 6.81 (d, J=7.9 Hz,1H), 6.87(s, 1H), 7.04(s, 1H), 7.18(s, 1H), 8.40(s, 1H). FAB HRMS [M+H]⁺calcd for C₂₅H₂₆N₆O₄S: 506.1736; found 507.1820.

M. 4-(9-Thia-1,5,7-triaza-fluoren-8-yl)-piperazine-1-carbothioic acid(benzo[1,3]dioxol-5-ylmethyl)-amide (10).

¹HNMR (CDCl₃, 300 MHZ) δ 4.07(s, 4H), 4.17(s, 4H), 4.72(d, J=4.5 Hz,2H), 5.88(s, 2H), 6.69(d, 1H), 6.75(d, 1H), 6.80(s, 1H), 7.43-7.47(m,1H), 8.65(s, 1H), 8.75(d, J=3.8 Hz, 2H). FAB HRMS [M+H]⁺ calcd forC₂₂H₂₀N₆O₂S₂: 464.1089; found 465.1167.

N. 4-(6,7-Dimethoxy-quinazolin-4-yl)-piperazine-1-carbothioic acid[4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide (11). (Scheme 15)

To a solution of 4-(1-piperazinyl)-6,7-dimethoxyquinazoline (200 mg,0.73 mmol) and pyridine (0.5 mL, 6.4 mmol) in dichloromethane (20 mL)was added a solution of compound 15 (Scheme 12) in dichloromethane (20mL) and this was stirred overnight. Methanol was added to quench excessthiophosgene, and the residue after removal of solvent was purified bysilica gel column chromatography eluting with 5%methanol/dichloromethane and further recrystallized fromdichloromethane/hexane to give 80 mg (20%) of compound 11.

¹HNMR (CDCl₃, 300 MHZ) δ 3.85(s, 4H), 3.98(s, 3H), 4.02(s, 3H), 4.11(s,4H), 6.98(m, 1H), 7.08(s, 1H), 7.32(d, 2H), 7.88(s, 1H), 8.00(d, J=6.7Hz, 2H), 8.62(d, 2H), 8.66(s, 1H). FAB HRMS [M+H]⁺ calcd forC₂₅H₂₆N₈O₄S₂: 566.1518; found 567.1597.

O. 6,7-dimethoxy-4-piperazin-1-yl-quinazoline

An analogous reaction to that described in Example 1, starting with4-Chloro-6,7-dimethoxy-quinazoline (32) in presence of piprazine andpyridine at refluxing temperature gave the title compound 33 as whitesolid.

P. 4-Chloro-6,7-dimethoxy-quinazoline

An analogous reaction to that described in Example 1, starting with6,7-Dimethoxy-3H-quinazolin-4-one (31) reacted with thionylchloride inpresence of DMF gave compound 32.

Q.7,8-Dimethoxy-4-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-5H-pyrimido[5,4-b]indole(12).

¹HNMR (DMSO-d6, 400 MHz) MS [+ve ESI] for C₂₁H₁₆N₆O₆S₂: found 613.0572(M+H)⁺.

R. 1-Benzo[1,3]dioxol-5-ylmethyl-3-[2-(6,7-dimethoxy-quinazolin-4ylamino)-pyrimidin-5yl]-thiourea(14).

¹HNMR (DMSO d6, 300 MHZ) δ 3.93(s, 3H), 3.96(s, 3H), 4.56(s, 2H),6.00(s, 2H), 6.84(d, 1H, J=7.9 Hz), 6.89(d, 1H, J=7.9 Hz), 6.95(s, 1H),7.25(s, 1H), 7.73(s, 1H), 8.45(s, 1H, br), 8.62(s, 2H), 9.5(s, 1H, br),10.59(s, 1H, br). FAB HRMS [M+H]⁺ calcd for C₂₃H₂₁N₇O₄S: 491.1376; found492.1454.

S. 4-(6,7-Dimethoxy-quinazolin-4-ylamino)-N-pyrimidin-2-yl-benzenesulfonamide (15)

¹HNMR (DMSO d6, 300 MHZ) δ 4.00(s, 6H), 7.08(m, 1H), 7.30(s, 1H,),7.96(d, 2H, J=8.7 Hz), 8.08((d, 2H, J=8.7 Hz), 8.15 (s, 1H), 8.53(d,2H), 8.85(s, 1H). FAB HRMS [M+H]⁺ calcd for C₂₀H₁₉N₆O₄S: 439.1178; found440.1180.

T. 4-(Benzo[4,5]furo[3,2-d]pyrimidin-4-ylamino-N-pyrimidin-2-yl-benzenesulfonamide (16).

¹HNMR (DMSO d6, 300 MHZ) 7.06(t, 1H), 7.58(t, 1H,), 7.79(t, 1H), 7.90(d,1H, J=8.4 Hz), 7.99(d, 2H, J=8.4 Hz), 8.16(d, 2H, J=8.9 Hz), 8.21(d, 1H,J=7.2 Hz), 8.53(d, 2H, J=4.9 Hz), 8.80 (s, 1H). FAB HRMS [M+H]⁺ calcdfor C₂₀H₁₅N₆O₃S: 419.4435; found 419.0935.

U.N-pyrimidin-2-yl-4(9-thia-1,5,7-triaza-fluoren-8ylamino)-benzenesulfonamide(17)

¹HNMR (DMSO d6, 300 MHZ) 7.01(t, 1H), 7.71(t, 1H,), 8.00(d, 2H, J=8.9Hz), 8.09(d, 2H, J=8.9 Hz), 8.48(d, 2H, J=5.2 Hz), 8.73(dd, 2H, J=6.8Hz), 8.88(m, 2H), 10.23 (s, 1H). FAB HRMS [M+H]⁺ calcd for C₁₉H₁₄N₇O₂S2:436.4979; found 436.0669.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A compound having the following structure:

or a stereoisomer, or pharmaceutically acceptable salt thereof, wherein:X is NH, S or O; Z is CH or N, with the proviso when X=NH then Z=CH; R₁and R₂ are the same or different and are independently hydrogen,hydroxyl, halo, —CN, —NO₂, —NH₂, —R, —OR, —SCH₃, —CF₃, —C(═O)OR or—OC(═O)R, where R is alkyl or substituted alkyl; R₃ is hydrogen, —NH₂,alkyl, —CN, or —NO₂; L₂ is selected from —NHCH₂—, —NH—, —C(═S)NH—,—NHC(═S)—, —C(═S)NHCH₂—, —NHC(═S)NH—, —NHC(═O)—, —NHC(═O)NH—; —S(═O)₂—;and Cycl₂ is:

where w is


2. The compound of claim 1, wherein L₂ is —C(═S)NH—.
 3. The compound ofclaim 1, wherein R₁ and R₂ are selected from —R or —OR where R is alkylor substituted alkyl, hydroxyl, halo, —CF₃, or —OC(═O)CH₃ is selectedfrom hydrogen or —NH₂.
 4. The compound of claim 1, wherein R₁ and R₂ areselected from —OCH₃, —Cl, or —CF₃, and R₃ is hydrogen.
 5. A compoundhaving the following structure:

or a stereoisomer, or pharmaceutically acceptable salt thereof, wherein:R₁ and R₂ are the same or different and are independently hydrogen,hydroxyl, halo, —CN, —NO₂, —NH₂, —R, —OR, —SCH₃, —CF₃, —C(═O)OR or—OC(═O)R, where R is alkyl or substituted alkyl; and R₃ is hydrogen,—NH₂, alkyl, —CN, or —NO₂.
 6. The compound of claim 5, wherein R₁ and R₂are selected from —R or —OR where R is alkyl or substituted alkyl,hydroxyl, halo, —CF₃, or —OC(═O)CH₃, and R₃ is hydrogen.
 7. The compoundof claim 5, wherein R₁ and R₂ are selected from —OCH₃, —CF₃, or —CI, andR₃ is hydrogen.
 8. A compound having the following structure:


9. A compound having the following structure:


10. A composition comprising a compound of any one of claims 1, 5, 8 and9 in combination with a pharmaceutically acceptable excipient.